Methods of treating or preventing inflammation and hypersensitivity with oxidative reductive potential water solution

ABSTRACT

Provided is a method for preventing or treating inflammation and associated states (e.g. infection, hypersensitivity, pain) by administering a therapeutically effective amount of an oxidative reduction potential (ORP) water solution that is stable for at least about twenty-four hours. The ORP water solution administered in accordance with the invention can be combined with one or more suitable carriers and can be administered in conjunction with one or more additional therapeutic agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 14/793,649, filed Jul. 7, 2015, which is a continuation of U.S.patent application Ser. No. 12/643,191, filed Dec. 21, 2009, which is acontinuation of U.S. patent application Ser. No. 11/656,087, filed Jan.22, 2007, which claims the benefit of U.S. Provisional Application Nos.60/760,635, filed Jan. 20, 2006; 60/760,567, filed Jan. 20, 2006;60/760,645, filed Jan. 20, 2006; and 60/760,557, filed Jan. 20, 2006;all of which are hereby incorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

Inflammation is a biological response that can result from a noxiousstimulus and is normally intended to remove that stimulus or ameliorateits effects. Although normally intended to promote survival,inflammation can cause damage to the host, especially in mammals. Thestimulus or insult initiating inflammation can be caused by endogenousfactors (e.g., an auto-antigen or irritating body fluid) or exogenousfactors (e.g., a foreign body or infectious agent).

Inflammation has been classified as “acute” and “chronic.” Acuteinflammation is typically of relatively short duration, lasting minutesto hours and, in some cases, a few days. Acute inflammation can becharacterized by the exudation of fluid and plasma proteins and theaccumulation of polymorphonuclear leukocytes (PMNLs) at the site of theinsult. Acute inflammation usually includes an increase in blood flow tothe area of the insult mediated by cellular molecules released inresponse to the insult. Increased vascular permeability also resultsfrom cellular mediators and leads to an accumulation of protein-richfluid. Important mediators of this increased blood flow and vascularpermeability include histamine from mast cells, serotonin andbradykinin.

In acute inflammation, PMNLs are also attracted to the area of insultand migrate out of the blood stream toward the insult. The PMNLs releasetoxic metabolites and proteinases that can cause tissue damage. Theseproteinases include proteins in the complement system, which can damagecell membranes and kallikreins which generate bradykinin. Acuteinflammation can undergo complete resolution, lead to the formation ofan abscess, result in scarring fibrosis or progress to chronicinflammation.

Chronic inflammation is of longer duration, lasting weeks to months, andpossibly years, in which tissue destruction and biological processesthat are intended to repair the injury are simultaneously ongoing.Chronic inflammation more typically involves lymphocytes and macrophagesand may also include a proliferation of blood vessels, fibrosis and/ornecrosis. Chronic inflammation can result from a number of conditionsincluding persistent infections, prolonged exposure to toxic agents, andautoimmune reactions. Chronic inflammation is often maintained by theproduction of cytokines by lymphocytes and macrophages at the site ofthe persistent insult. Chronic inflammation can result in permanenttissue damage or complete healing.

Hypersensitivity generally refers to inflammation that causes damage tothe host, in which the damage outweighs the benefit to the host.Hypersensitivity can result in significant pathology including, e.g.,anaphylaxis, transplant rejection, and autoimmune diseases. The mostcommon type of hypersensititvity is allergy.

Independently of the inducing factor -and the length of the exposure- aninflammatory reaction is mediated by a varied number and type of cellsand molecules, the later including cytokines, growth factors, clottingfactors, enzymes, neurotransmitters and complement proteins, amongothers. These molecules are primarily secreted by fibroblasts,endothelial and infiltrating cells (e.g. macrophages, lymphocytes, mastcells, polymorphonuclear cells, etc), and local nerves in response tothe insulting agent. The mixture and amount of cytokines thereinreleased will depend on the type, concentration and exposure time of theinducing agent. Therefore, these proteins could mediate from an acutelocal inflammatory reaction to systemic life-threatening responses (e.g.acute systemic inflammatory response syndrome, SIRS; multiple organfailure as in septic shock; anaphylaxis, etc). In chronic inflammatoryprocesses, the cytokines continuously recruit more and more infiltratingcells that generate, for example, granulomas, induration of the tissues,and encapsulated abscesses. In any case, proteins secreted during aninflammatory process are central players in the grade and persistence ofthe final reaction.

Stimulation of the aforementioned cells by the induction agent leads toa cascade of intracellular signaling events that ultimately result inproduction and secretion of cytokines and other inflammatory mediatorsthat constitute the pro-inflammatory response. While thepro-inflammatory response is crucial for effective clearance of thepathogen or allergen, the inflammatory mediators produced cause tissuedamage and inflammation. Hence, a balance needs to be maintained betweenthe activation and down-regulation of this response in order to avoidsevere tissue damage (Cohen, J. The immunopathogenesis of sepsis. Nature2002 420, 885-891). Dysregulation of this response could induce localdamage (e.g. lung fibrosis) or could lead to potentially lethalconditions like septic shock and systemic inflammatory response syndrome(SIRS) as previously mentioned. Thus, microbes allergens, endotoxins,and many other molecules induce the production of pro-inflammatorymediator proteins by different cells in the human body. The combinedeffects of all these molecules in living tissues could mediate changesin the clotting system, wound healing process, anti-microbial activity,antibody production and the perception of pain, among many otherreactions.

The systemic inflammatory response syndrome (SIRS), a syndrome thatencompasses the features of systemic inflammation without end-organdamage or identifiable bacteremia. SIRS is separate and distinct fromsepsis, severe sepsis or septic shock. The key transition from SIRS tosepsis is the presence of an identified pathogen in the blood. Thepathophysiology of SIRS includes, but is not limited to, complementactivation, cytokine and arachidonic acid metabolites secretion,stimulated cell-mediated immunity, activation of the clotting cascades,and humoral immune mechanisms. Clinically SIRS is characterized bytachycardia, tachypnea, hypotension, hypoperfusion, oliguria,leukocytosis or leukopenia, pyrexia or hypothermia, metabolic acidosis,and the need for volume support. SIRS may affect all organ systems andmay lead to multiple organ dysfunction syndrome (MODS). Thus, even inearly stages (i.e. SIRS), there is accumulation of pro-inflammatorycytokines at the primary site of inflammation and in the blood that cancontribute to the establishment of multi-organ failure and death.

Typically, inflammation is treated with steroidal or non-steroidalanti-inflammatory drugs. However, conventional anti-inflammatory therapysuffers from several drawbacks, e.g., systemic toxicity, allergicreactions, insulin resistance, hypertension, cardiac toxicity, renaltoxicity, various coagulopathies and gastric erosions. Accordingly,there is a need for mild, yet safe and effective methods for treating orpreventing inflammation. The present invention provides such methods.These and other advantages of the invention, as well as additionalinventive features, will be apparent from the description of theinvention provided herein.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a method of preventing or treatinginflammation in a patient by administering to the patient atherapeutically effective amount of an oxidative reductive potential(ORP) water solution, wherein the solution is stable for at leasttwenty-four hours. The method of the present invention can be used inthe treatment of inflammation resulting from a variety of causativefactors, e.g., allergic reaction, autoimmune reaction, infection,contact with one or more inflammation-causing substances, andcombinations of such causative factors.

The method of the present invention can further include administeringthe ORP water solution in conjunction with one or more therapeuticagents, e.g., one or more compounds selected from the group consistingof antibiotics, anti-viral agents, anti-inflammatory agents, andcombinations thereof. Administering such therapeutic agents inconjunction with the ORP water solution includes administering one ormore of such agents, e.g., prior to, during (e.g., contemporaneously, byco-administration or in combination with), or following administrationof the ORP water solution.

The ORP water solution can be administered by any suitable route inaccordance with the present invention, e.g., by delivering the ORP watersolution topically or parenterally, so as to contact a therapeuticallyeffective amount of the ORP water solution with one or more affectedtissues, which may reside inside or outside of the body. Accordingly,the invention provides a method wherein the ORP water solution isadministered to one or more tissues, e.g., nasal, sinus, pharyngeal,tracheal, pulmonary, esophageal, gastric, intestinal, mesothelial,peritoneal, synovial, urinary bladder, urtheral, vaginal, uterine,fallopian, pancreatic, nervous, oral, cutaneous, and subcutaneous. TheORP water solution can be administered in any suitable form inaccordance with the present invention, e.g., as a liquid, spray, mist,aerosol or steam, and, if desired, can be combined with one or moresuitable carriers, e.g., vehicles, adjuvants, excipients, diluents, andthe like.

The ORP water solution administered in accordance with the presentinvention can be contained within a sealed container and is stable forat least twenty-four hours. The ORP water solution administered inaccordance with the invention can be produced by electrolysis, andpreferably comprises a mixture of anode water and cathode water, whichcontains one or more species, including, e.g., reactive species, ionicspecies, radical species, precursors thereof and combinations thereof.The ORP water solution administered in accordance with the inventionexhibits potent anti-inflammatory activity, yet is virtually free oftoxicity to normal tissues and normal eukaryotic cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a three-chambered electrolysis cell for producing anexemplary ORP water solution.

FIG. 2 illustrates a three-chambered electrolysis cell and depicts ionicspecies that are believed to be generated during the production process.

FIG. 3 is a schematic flow diagram of a process for producing anexemplary ORP water solution.

FIG. 4A-4C depict a graphical comparison of cell viability, apoptosisand necrosis in human diploid fibroblasts (HDFs) treated with anexemplary ORP water solution (MCN) versus hydrogen peroxide (HP).

FIG. 5 is a graphical comparison of the levels of8-hydroxy-2′-deoxiguanosine (8-OHdG) adducts in HDFs treated with anexemplary ORP water solution (MCN) versus 500 μM hydrogen peroxide (HP).

FIG. 6 illustrates cellular senescence demonstrated by β-galactosidaseexpression in HDFs after chronic exposure to low concentrations of anexemplary ORP water solution (MCN) versus hydrogen peroxide (HP).

FIG. 7 illustrates the effect on degranulation of antigen-activated mastcells treated with various concentrations of an exemplary ORP watersolution (MCN).

FIG. 8 comparatively illustrates the effect on degranulation ofantigen-activated mast cells treated with cromoglycate.

FIG. 9 illustrates the effect on degranulation of antigen-activated andcalcium ionophore (A23187)-activated mast cells treated with variousconcentrations of an exemplary ORP water solution (MCN).

FIG. 10A-10B are RNAse protection assays illustrating cytokine mRNAlevels after antigen challenge in control versus ORP watersolution-treated mast cells.

FIG. 11 is a graphical comparison of TNF-α secretion byantigen-activated mast cells treated with various concentrations of anexemplary ORP water solution (MCN).

FIG. 12 is a graphical comparison of MIP1-α secretion byantigen-activated mast cells treated with various concentrations of anexemplary ORP water solution (MCN).

FIG. 13 is a graphical comparison of IL-6 secretion by antigen-activatedmast cells treated with various concentrations of an exemplary ORP watersolution (MCN).

FIG. 14 is a graphical comparison of IL-13 secretion byantigen-activated mast cells treated with various concentrations of anexemplary ORP water solution (MCN).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of preventing or treatinginflammation in a patient, which method comprises administering to thepatient a therapeutically effective amount of an oxidative reductivepotential (ORP) water solution (also known as super-oxidized water(SOW)), wherein the solution is stable for at least about twenty-fourhours. The method of the present invention can be used for treating orpreventing (e.g., inhibiting the onset of, inhibiting the escalation of,decreasing the likelihood of) acute inflammation and chronicinflammation, including hypersensitivity such as, e.g., in allergies.The inflammation and hypersensitivity treatable or preventable inaccordance with the method of the present invention can includeinflammation that results from, e.g., contact with a noxious stimulus,injury, infection, autoimmune reaction, hypersensitivity, and allergicreaction, including allergic reactions associated with cellularhistamine and pro-inflammatory cytokine release.

Surprisingly, it has been found that the ORP water solution administeredin accordance with the invention is a highly effective inhibitor of mastcell degranulation, one of the primary inflammation and allergy-causingbiological cascades. The ORP water solution administered in accordancewith the invention inhibits degranulation of mast cells regardless ofwhether they are activated with an antigen or a calcium ionophore. Alsosurprisingly, it has been found that the ORP water solution administeredin accordance with the present invention non-selectively inhibits thesecretion of pro-inflammatory cytokines in mast cells. For example, theORP water solution of the present invention can inhibit the secretionof, e.g., TNF-α, MIP1-α, IL-6, and IL-13 in mast cells. It is believedthat the ORP water solution administered in accordance with theinvention also can inhibit the secretion of pro-inflammatory cytokinesin other cytokine-secreting cells including, but not limited to,macrophages, monocytes, lymphocytes, macrophages, PMN, fibroblasts andendothelial cells. These findings demonstrate that the ORP wateradministered in accordance with the present invention should exhibitbroad anti-inflammatory efficacy.

The ORP water solution administered in accordance with the inventionpreferably inhibits mast cell degranulation by more than about 50%relative to untreated mast cells, more preferably by more than about 80%relative to untreated mast cells, still more preferably by more thanabout 90% relative to untreated mast cells, and even more preferably bymore than about 95% relative to untreated mast cells, when contactedwith the ORP water solution for up to about 30 minutes, more preferablyup to about 15 minutes, and still more preferably up to about 5 minutes.

The ORP water solution administered in accordance with the inventionalso preferably inhibits the secretion of TNF-α by more than about 50%,more preferably by more than about 60%, still more preferably by morethan about 70%, and even more preferably by more than about 85%. Inaddition, the ORP water solution administered in accordance with theinvention also preferably inhibits the secretion of MIP1-α by more than25%, more preferably by more than about 50%, and still more preferablyby more than about 60%. Further, the ORP water solution administered inaccordance with the invention also preferably inhibits the secretion ofIL-6 and/or IL-13 by more than 25%, more preferably by more than about50%, and still more preferably by more than about 60%. In accordancewith the method of the invention, secretion of these and that of othercytokines, can be therapeutically inhibited down to certain % by theadministration of the ORP water solution alone or in combination with adiluent (e.g., water), by increasing the concentration of the componentsof the ORP water solution, by utilizing special delivery systems and/orby increasing the exposure time. For instance, cytokine secretion can betherapeutically inhibited by administering compositions in which the ORPwater solution is diluted, e.g., by a ratio of up to about 50%(vol./vol.) ORP water solution/diluent, by a ratio of up to about 25%(vol./vol.) ORP water solution/diluent, by a ratio of up to about 10%(vol./vol.) ORP water solution/diluent, by a ratio of up to about 5%(vol./vol.) ORP water solution/diluent, or even by a ratio of up toabout 1% (vol./vol.) ORP water solution/diluent.

The method of the present invention can be used for treating orpreventing cell-mediated inflammation, which results from an autoimmunereaction, including, but not limited to, SLE, autoimmune thyroiditis,sarcoidosis, inflammatory bowel disease, rheumatoid arthritis, rheumaticfever, psoriasis, pemphigus, erythema multiforme, other bullous diseasesof the skin, and atopias. The method of the invention can be used fortreating or preventing inflammation, which results from infection,allergens, foreign bodies, and autoimmune processes. The method of theinvention can also be used for treating or preventing inflammation,which results from infection, e.g., from an infection by one or moremicroorganisms selected from the group consisting of viruses, bacteria,and fungi, including hypersensitivity and autoimmune-mediatedinflammation resulting from infection.

The method of the present invention can be used for treating orpreventing inflammation associated with an upper respiratory condition.When the inflammation is associated with an upper respiratory condition,the ORP water solution is preferably administered to the upper airway,e.g., as a spray, mist, aerosol or steam, so as to contact one or moreupper airway tissues affected by the condition. Any suitable method canbe employed for delivering the ORP water solution to the upper airway soas to treat or prevent one or more upper respiratory conditions inaccordance with the present invention, including one or more routes ofadministration described herein.

The method of the present invention can be used for preventing ortreating inflammation affecting one or more upper respiratory airwaytissues (e.g., nasal tissue, sinus tissue) or lung tissues. Suchconditions can include, for example, sinusitis (e.g., rhinosinusitis,acute sinusitis, chronic sinusitis, and the like), pharyngitis, asthma,and the like, which are preventable or treatable with the ORP solutionadministered in accordance with the invention.

Chronic sinusitis typically refers to inflammation of the sinuses thatcontinues for at least 3 weeks, but the inflammation can (and oftendoes) continue for months or even years. Allergies are frequentlyassociated with chronic sinusitis. In addition, patients with asthmahave a particularly high frequency of chronic sinusitis. Inhalation ofairborne allergens (substances that provoke an allergic reaction), suchas dust, mold, and pollen, often set off allergic reactions (e.g.,allergic rhinitis) that, in turn, may contribute to sinusitis(particularly rhinosinusitis or rhinitis). People who are allergic tofungi can develop a condition called “allergic fungal sinusitis.” Dampweather or pollutants in the air and in buildings also can affect peoplesubject to chronic sinusitis.

Like acute sinusitis, chronic sinusitis is more common in patients withimmune deficiency or abnormalities of mucus secretion or movement (e.g.,immune deficiency, HIV infection, cystic fibrosis, Kartagener'ssyndrome). In addition, some patients have severe asthma, nasal polyps,and severe asthmatic responses to aspirin and aspirin-like medications(so-called non-steroidal anti-inflammatory drugs, or NSAIDs). Theselatter patients have a high frequency of chronic sinusitis.

A doctor can diagnose sinusitis by medical history, physicalexamination, X-rays, and if necessary, MRIs or CT scans (magneticresonance imaging and computed tomography). After diagnosing sinusitisand identifying a possible cause, a doctor can prescribe a course oftreatment that will reduce the inflammation and relieve the symptoms.Treating acute sinusitis typically requires re-establishing drainage ofthe nasal passages, controlling or eliminating the source of theinflammation, and relieving the pain. Doctors generally recommenddecongestants to reduce the congestion, antibiotics to control abacterial infection, if present, and pain relievers to reduce the pain.

When treatment with drugs fails, surgery may be the only alternative fortreating chronic sinusitis, e.g., removal of adenoids, removal of nasalpolyps, repair of a deviated septum, endoscopic sinus surgery, and thelike. It is believed that the administration of ORP water in accordancewith the method of the present invention can be used for treatingchronic sinusitis and inflammation associated therewith as analternative to potentially avoid more aggressive therapies, such asantibiotics and surgery.

With regard to pharyngitis, it is estimated that worldwide, 1 to 2% ofall visits to doctors' offices, clinics and emergency rooms are becauseof pharyngitis. In the United States and Mexico, pharyngitis andtonsillitis is believed to account for about 15 and 12 millionconsultations per year, respectively. These cases are typically causedby various bacteria and viruses. Also, pharyngitis and tonsillitiscaused by group A β-hemolytic Streptococcus can significantly raise therisk of rheumatic fever in poor populations; however it is believed thatonly 5 to 15% of pharyngitis cases are caused by this bacterium, andthat the rest of the acute cases are due to bacteria and viruses oflittle epidemiological relevance. The latter cases tend to beself-limiting in a few days and do not leave sequelae.

It has been verified that a great number of doctors worldwide prescribeantibiotics indiscriminately for acute pharyngitis. This occurs in adaily practice, often because patients tend to request powerfulantibiotics. Unfortunately, it is difficult to establish an accuratediagnosis of streptococcal pharyngitis/tonsillitis clinically and thecost/benefit ratio of treating acute pharyngitis/tonsillitis withantibiotics is questionable.

It is believed that the method of the present invention provides a safe,efficacious and cost-effective adjuvant therapy for the treatment orprevention of acute pharyngitis and/or tonsillitis due to bacteriaand/or viruses. The empirical treatment of acute pharyngitis/tonsillitismay begin with administering an ORP water solution in accordance withthe present invention, and, depending on evolution or the result of therapid test for Streptococcus, antibiotics may be initiated from 48-72hours thereafter only if needed. The method of the present invention maythus allow the use of antibiotics to be deferred and, at the same time,reduce the symptomatology of the patient and accelerate the patient'srecovery if the pharyngitis/tonsillitis is not from group AStreptococcus. The adjuvant use of an ORP water solution of the presentinvention with antibiotics for the treatment of streptococcalpharyngitis/tonsillitis also may shorten the period of clinical responseand decrease the incidence of recurrences.

The method of the present invention also can be used for treating orpreventing inflammation associated with hypersensitivity. Historically,hypersensitivity reactions have been classified as one of four types,from which significant disease can result. The ORP water solutionadministered in accordance with the invention can be used to treatand/or prevent (e.g., inhibit the onset of, inhibit the escalation of ordecrease the likelihood of) one or more of such reactions. Type Ihypersensitivity typically results from the combination of an antigenwith an antibody bound to a mast cell or basophil. Type I reactionsoccur within minutes of exposure to the antigen in individuals who havebeen previously sensitized to the antigen. In humans, Type I reactionsare mediated by IgE which has high affinity Fc receptors on mast cellsand basophils.

Mast cells' role in Type I hypersensitivity is especially importantbecause they reside in tissues under the epithelial surface near bloodvessels and nerves. Multiple clinical symptoms observed in atopicdermatitis, allergic rhinitis and atopic asthma are produced byIgE-antigen stimulation of mast cells located in distinct affectedtissues. The currently accepted view of the pathogenesis of atopicasthma is that allergens initiate the process by triggering IgE-bearingpulmonary mast cells (MCs) to release mediators such as histamine,leukotrienes, prostaglandins, kininis, platelet activating factor (PAF),etc. in the so-called early phase of the reaction (see Kumar et al.,Robbins & Cotran Pathologic Basis of Disease, 2004, pp. 193-268, whichis hereby incorporated by reference). In turn, these mediators inducebronchoconstriction and enhance vascular permeability and mucusproduction. According to this model, following mast cell activation inthe late phase, those cells secrete various cytokines, including tumornecrosis factor alpha (TNF-α), IL-4, IL-5 and IL-6, which participate inthe local recruitment and activation of other inflammatory cells such aseosinophils, basophils, T lymphocytes, platelets and mononuclearphagocytes. These recruited cells, in turn, contribute to thedevelopment of an inflammatory response that may then become autonomousand aggravate the asthmatic symptoms. This late phase responseconstitutes a long term inflammatory process which will induce changesin surrounding tissues (Kumar et al., pp. 193-268). Clinically, Type Ireactions can have local effects such as allergic rhinitis, or systemiceffects as is found in anaphylaxis which manifests with itching, hives,respiratory distress, and circulatory collapse.

Type II hypersensitivity is mediated by antibodies directed to antigenson the surfaces of cells and in the extracellular space. Theseantibodies can direct cell lysis or result in opsonization of the targetmolecules (preparation for phagocytosis by other cells). Alternatively,the antibodies can be directed to and activate cell surface receptors.Conditions resulting from Type II reactions include transfusionreactions, Graves disease (thyrotoxicosis), drug reactions, perniciousanemia, and acute rheumatic fever. In rheumatic fever the antibodies areformed against Streptococcal antigens but, cross-react with humantissues such as heart valves.

Type III hypersensitivity is caused by immune complexes, which arecombinations of antibodies and other host immune system proteins, mosttypically complement proteins. It is the normal function of antibodiesto bind and active complement. However, when the resultingmacromolecular immune complexes are not adequately processed, they canlead to persistent tissue damage. Macrophages and PMNLs can be activatedby immune complexes and lead to the release of toxic chemicals by thesecells. Immune complex reactions can be local and may result inconditions such as, e.g., the arthus reaction or cause systemic diseasesuch as serum sickness or some of the aspects of systemic lupuserythematous (SLE).

Type IV hypersensitivity is cell mediated and is sometimes calleddelayed-type hypersensitivity. Type IV hypersensitivity is mediated by Tlymphocytes and often results in the formation of a granulomatousreaction. In a granulomatous reaction, a form of macrophage called anepitheloid cells attempts to, but fails, to digest an antigen. Theantigen's persistence leads to the release of cytokines that attractadditional lymphocytes resulting in chronic foci of inflammation. Thefoci have high concentrations of cyotoxic T-lymphocytes which releasegranzymes and perforins which are toxic to adjacent cells. Type IVhypersensitivity is a prominent component of autoimmune diseases suchas, e.g., Sjogrren's Syndrome, Sarcoidosis, and contact dermatitis.

Pathologic states can combine different types of hypersensitivityreactions. In autoimmune diseases host antigens stimulatehypersensitivity with serious consequences for the host. For example, inSLE host antigens induce Type II reactions against blood cells whileType III reactions lead to blood vessel and renal glomerular damage. Inaddition, hypersensitivity reactions are also seen in iatragenicconditions such as drug reactions and transplant rejection. Transplantrejection includes components of Type II and Type IV hypersensivity.Accordingly, ORP water solution used in accordance with the invention intransplantable organs or cells could greatly reduced the possibility ofbeing rejected by the host.

It has been found that the ORP water solution administered in accordancewith the invention is virtually free of toxicity to normal tissues andnormal mammalian cells. The ORP water solution administered inaccordance with the invention causes no significant decrease in theviability of eukaryotic cells, no significant increase in apoptosis, nosignificant acceleration of cell aging and/or no significant oxidativeDNA damage in mammalian cells. The non-toxicity is particularlyadvantageous, and perhaps even surprising, given that the disinfectingpower of the ORP water solution administered in accordance with theinvention is roughly equivalent to that of hydrogen peroxide, yet issignificantly less toxic than hydrogen peroxide is to normal tissues andnormal mammalian cells. These findings demonstrate that the ORP watersolution administered in accordance with the present invention is safefor use, e.g., in mammals, including humans.

For the ORP water solution administered in accordance with theinvention, the cell viability rate is preferably at least about 65%,more preferably at least about 70%, and still more preferably at leastabout 75% after an about 30 minute exposure to the ORP water solution.In addition, the ORP water solution administered in accordance with theinvention preferably causes only up to about 10% of cells, morepreferably only up to about 5% of cells, and still more preferably onlyup to about 3% of cells to expose Annexin-V on their cellular surfaceswhen contacted with the ORP water solution for up to about thirtyminutes or less (e.g., after about thirty minutes or after about fiveminutes of contact with the ORP water solution).

Further, the ORP water solution administered in accordance with theinvention preferably causes less than about 15% of cells, morepreferably less than about 10% of cells, and still more preferably lessthan about 5% of cells to express the SA-β-galactosidase enzyme afterchronic exposure to the OPR water solution. The ORP water solutionadministered in accordance with the invention preferably causes causedthe same fraction of the oxidative DNA adduct formation caused by salinesolution, e.g., less than about 20% of the oxidative DNA adductformation, less than about 10% of the oxidative DNA adduct formation, orabout 5% or less of the oxidative DNA adduct formation normally causedby hydrogen peroxide in cells treated under equivalent conditions.

The ORP water solution administered in accordance with the inventionproduces no significant RNA degradation. Accordingly, RNA extracted fromhuman cell cultures after an about 30 minutes exposure to the ORP watersolution or r at about 3 hours after an about 30 minute-exposure, andanalyzed by denaturing gel electrophoresis, will typically show nosignificant RNA degradation and will typically exhibit two discreetbands corresponding to the ribosomal eukaryotic RNAs (i.e. 28S and 18S)indicating that the ORP water solution administered in accordance withthe invention leaves the RNA substantially intact. Similarly, RNAextracted from human cell cultures after about 30 minutes of exposure tothe ORP water solution or after about 3 hours of exposure, can besubjected reverse transcription and amplification (RT-PCR) of theconstitutive human GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) geneand result in a strong GAPDH band on gel electrophoresis of the RT-PCRproducts. By contrast, cells treated with HP for a similar period showsignificant RNA degradation and little if any GAPDH RT-PCR product.

The ORP water solution used in accordance with the present invention canbe administered using any suitable method of administration known in theart. For instance, the ORP water solution can be administeredparenterally, endoscopically or directly to the surface of any affectedbiological tissue, e.g., to the skin and/or one or more mucosalsurfaces. Parenteral administration can include using, for example,administering the ORP water solution intramuscularly, subcutaneously,intravenously, intra-arterially, intrathecally, intravesically or into asynovial space. Endoscopic administration of the ORP water solution caninclude using, e.g., bronchoscopy, colonoscopy, sigmoidoscopy,hysterscopy, laproscopy, athroscopy, gastroscopy or a transurethralapproach. Administering the ORP water solution to a mucosal surface caninclude, e.g., administration to a nasal, oral, tracheal, bronchial,esophageal, gastric, intestinal, peritoneal, urethral, vesicular,urethral, vaginal, uterine, fallopian, and synovial mucosal surface.

Parenteral administration also can include administering the ORP watersolution used in accordance with the invention intravenously,subcutaneously, intramuscularly, or intraperitoneally. The ORP watersolution of the present invention can be administered intravenously asdescribed, e.g., in U.S. Pat. Nos. 5,334,383 and 5,622,848 (herebyincorporated by reference), which describe methods of treating viralmyocarditis, multiple sclerosis, and AIDS via intravenous administrationof ORP water solutions. Other applications include the treatment of anyhypersensitivity and infectious processes, as mentioned above.

The ORP water solution used in accordance with the invention can beadministered topically, e.g., as a liquid, spray, mist, aerosol or steamby any suitable process, e.g., by aerosolization, nebulization oratomization. The ORP solution of the present invention can beadministered to the upper airway as a steam or a spray. When the ORPwater solution is administered by aerosolization, nebulization oratomization, it is preferably administered in the form of dropletshaving a diameter in the range of from about 0.1 micron to about 100microns, preferably from about 1 micron to about 10 microns. In oneembodiment, the method of the present invention includes administeringthe ORP water solution in the form of droplets having a diameter in therange of from about 1 micron to about 10 microns to one or more mucosaltissues, e.g., one or more upper respiratory tissues and/or lungtissues.

Methods and devices, which are useful for aerosolization, nebulizationand atomization, are well known in the art. Medical nebulizers, forexample, have been used to deliver a metered dose of a physiologicallyactive liquid into an inspiration gas stream for inhalation by arecipient. See, e.g., U.S. Pat. No. 6,598,602 (hereby incorporated byreference). Medical nebulizers can operate to generate liquid droplets,which form an aerosol with the inspiration gas. In other circumstancesmedical nebulizers may be used to inject water droplets into aninspiration gas stream to provide gas with a suitable moisture contentto a recipient, which is particularly useful where the inspiration gasstream is provided by a mechanical breathing aid such as a respirator,ventilator or anaesthetic delivery system.

An exemplary nebulizer is described, for example, in WO 95/01137, whichdescribes a hand held device that operates to eject droplets of amedical liquid into a passing air stream (inspiration gas stream), whichis generated by a recipient's inhalation through a mouthpiece. Anotherexample can be found in U.S. Pat. No. 5,388,571 (hereby incorporated byreference), which describes a positive-pressure ventilator system whichprovides control and augmentation of breathing for a patient withrespiratory insufficiency and which includes a nebulizer for deliveringparticles of liquid medication into the airways and alveoli of the lungsof a patient. U.S. Pat. No. 5,312,281 (hereby incorporated by reference)describes an ultrasonic wave nebulizer, which atomizes water or liquidat low temperature and reportedly can adjust the size of mist. Inaddition, U.S. Pat. No. 5,287,847 (hereby incorporated byreference)describes a pneumatic nebulizing apparatus with scalable flowrates and output volumes for delivering a medicinal aerosol to neonates,children and adults. Further, U.S. Pat. No. 5,063,922 (herebyincorporated by reference) describes an ultrasonic atomizer. The ORPwater solution also may be dispensed in aerosol form as part of aninhaler system for treatment of infections in the lungs and/or airpassages or for the healing of wounds in such parts of the body.

For larger scale applications, a suitable device may be used to dispersethe ORP water solution into the air including, but not limited to,humidifiers, misters, foggers, vaporizers, atomizers, water sprays, andother spray devices. Such devices permit the dispensing of the ORP watersolution on a continuous basis. An ejector which directly mixes air andwater in a nozzle may be employed. The ORP water solution may beconverted to steam, such as low pressure steam, and released into theair stream. Various types of humidifiers may be used such as ultrasonichumidifiers, stream humidifiers or vaporizers, and evaporativehumidifiers. The particular device used to disperse the ORP watersolution may be incorporated into a ventilation system to provide forwidespread application of the ORP water solution throughout an entirehouse or healthcare facility (e.g., hospital, nursing home, etc.).

In accordance with the invention, the ORP water solution can beadministered alone or in combination with one or more pharmaceuticallyacceptable carriers, e.g., vehicles, adjuvants, excipients, diluents,combinations thereof, and the like, which are preferably compatible withone or more of the species that exist in the ORP water solution. Oneskilled in the art can easily determine the appropriate formulation andmethod for administering the ORP water solution used in accordance withthe present invention. Any necessary adjustments in dose can be readilymade by a skilled practitioner to address the nature and/or severity ofthe condition being treated in view of one or more clinically relevantfactors, such as, e.g., side effects, changes in the patient's overallcondition, and the like.

For example, the ORP water solution can be formulated by combining ordiluting the ORP water solution with up to about 25% (wt./wt. orvol./vol.) of a suitable carrier, up to about 50% (wt./wt. or vol./vol.)of a suitable carrier, up to about 75% (wt./wt. or vol./vol.) of asuitable carrier, up to about 90% (wt./wt. or vol./vol.) of a suitablecarrier, up to about 95% (wt./wt. or vol./vol.) of a suitable carrier,or even with up to about 99% (wt./wt. or vol./vol.) or more of asuitable carrier. Suitable carriers can include, e.g., water (e.g.,distilled water, sterile water, e.g., sterile water for injection,sterile saline and the like). Suitable carriers also can include one ormore carriers described in U.S. patent application Ser. No. 10/916,278(hereby incorporated by reference). Exemplary formulations can include,e.g., solutions in which the ORP water solution is diluted with sterilewater or sterile saline, wherein the ORP water solution is diluted by upto about 25% (vol./vol.), by up to about 50% (vol./vol.), by up to about75% (vol./vol.), by up to about 90% (vol./vol.), by up to about 95%(vol./vol.), or by up to 99% (vol./vol.) or more of a suitable carrier.

The ORP water solution administered in accordance with the invention canfurther be combined with (or be administered in conjunction with) one ormore additional therapeutic agents, e.g., one or more active compoundsselected from the group consisting of antibacterial agents (e.g.,antibiotics), anti-viral agents, anti-inflammatory agents, andcombinations thereof.

The therapeutically effective amount administered to the patient, e.g.,a mammal, particularly a human, in the context of the present inventionshould be sufficient to affect a therapeutic or prophylactic response inthe patient over a reasonable time frame. The dose can be readilydetermined using methods that are well known in the art. One skilled inthe art will recognize that the specific dosage level for any particularpatient will depend upon a variety of potentially therapeuticallyrelevant factors. For example, the dose can be determined based on thestrength of the particular ORP water solution employed, the severity ofthe condition, the body weight of the patient, the age of the patient,the physical and mental condition of the patient, general health, sex,diet, the frequency of applications, and the like. The size of the dosealso can be determined based on the existence, nature, and extent of anyadverse side effects that might accompany the administration of aparticular ORP water solution. It is desirable, whenever possible, tokeep adverse side effects to a minimum.

Factors, which can be taken into account for a specific dosage caninclude, for example, bioavailability, metabolic profile, time ofadministration, route of administration, rate of excretion, thepharmacodynamics associated with a particular ORP water solution in aparticular patient, and the like. Other factors can include, e.g., thepotency or effectiveness of the ORP water solution with respect to theparticular condition to be treated, the severity of the symptomspresented prior to or during the course of therapy, and the like. Insome instances, what constitutes a therapeutically effective amount alsocan be determined, in part, by the use of one or more of the assays,e.g., bioassays, which are reasonably clinically predictive of theefficacy of a particular ORP water solution for the treatment orprevention of a particular condition.

The ORP water solution used in accordance with the present invention canbe administered, alone or in combination with one or more additionaltherapeutic agents, to a patient, e.g., a human, e.g., to treat anexisting condition. The ORP water solution of the present invention alsocan be administered prophylactically, alone or in combination with oneor more additional therapeutic agents, to a patient, e.g., a human, thathas been exposed to one or more causative agents associated with thecondition. For example, the ORP water solution can be suitablyadministered prophylactically to a patient that has been exposed to oneor more inflammation-causing microorganisms (e.g., infections, viruses,bacteria and/or fungi) -or hypersensitivity epitope or allergen- toinhibit or decrease the likelihood of inflammation (and even infection)associated with the microorganism or epitope in a patient, or decreasethe severity of an inflammation (and even infection or allergy) thatdevelops as a result of such exposure.

One skilled in the art will appreciate that suitable methods ofadministering the ORP water solution used in accordance with the presentinvention are available, and, although more than one route ofadministration can be used, a particular route can provide a moreimmediate and more effective reaction than another route. Thetherapeutically effective amount can be the dose necessary to achieve an“effective level” of the ORP water solution in an individual patient,independent of the number of applications a day. The therapeuticallyeffective amount can be defined, for example, as the amount required tobe administered to an individual patient to achieve a blood level,tissue level, and/or intracellular level of the ORP water solution (orone or more active species contained therein) to prevent or treat thecondition in the patient.

When the effective level is used as a preferred endpoint for dosing, theactual dose and schedule can vary depending, for example, uponinterindividual differences in pharmacokinetics, distribution,metabolism, and the like. The effective level also can vary when the ORPwater solution is used in combination with one or more additionaltherapeutic agents, e.g., one or more anti-infective agents, one or more“moderating,” “modulating” or “neutralizing agents,” e.g., as describedin U.S. Pat. Nos. 5,334,383 and 5,622,848 (hereby incorporated byreference), one or more anti-inflammatory agents, and the like.

An appropriate indicator can be used for determining and/or monitoringthe effective level. For example, the effective level can be determinedby direct analysis (e.g., analytical chemistry) or by indirect analysis(e.g., with clinical chemistry indicators) of appropriate patientsamples (e.g., blood and/or tissues). The effective level also can bedetermined, for example, by direct or indirect observations such as,e.g., the concentration of urinary metabolites, changes in markersassociated with the condition (e.g., viral count in the case of a viralinfection), histopathology and immunochemistry analysis, positivechanges in image analysis (e.g. X ray, CT scan, NMR, PET, etc), nuclearmedicine studies, decrease in the symptoms associated with theconditions, and the like.

Conventional ORP water solutions have an extremely limited shelf-life,usually only a few hours. As a result of this short lifespan, usingconventional ORP water solutions requires the production to take placein close proximity to the point of use. From a practical standpoint,this means that the facility, e.g., a healthcare facility such as ahospital, must purchase, house and maintain the equipment necessary toproduce conventional ORP water solution. Additionally, conventionalmanufacturing techniques have not been able to produce sufficientcommercial-scale quantities to permit widespread use, e.g., as a generaldisinfecting agent for healthcare facilities.

Unlike conventional ORP water solutions, the ORP water solutionadministered in accordance with the invention is stable for at leastabout twenty-hours after its preparation. In addition, the ORP watersolution administered in accordance with the invention is generallyenvironmentally safe and, thus, avoids the need for costly disposalprocedures. Preferably, the ORP water solution administered inaccordance with the invention is stable for at least about one week(e.g., one week, two weeks, three weeks, four weeks or more.), and morepreferably at least about two months. Still more preferably, the ORPwater solution administered in accordance with the invention is stablefor at least about six months. Even more preferably, the ORP watersolution administered in accordance with the invention is stable for atleast about one year, and most preferably is stable for more than aboutone year, e.g., at least about two years or at least about three years.

Stability can be measured based on the ability of the ORP water solutionto remain suitable for one or more uses, for example, inhibiting mastcell degranulation, inhibiting cytokine secretion, decontamination,disinfection, sterilization, anti-microbial cleansing, and woundcleansing, for a specified period of time after its preparation undernormal storage conditions (e.g., room temperature). The stability of theORP water solution administered in accordance with the invention alsocan be measured by storage under accelerated conditions, e.g., fromabout 30° C. to about 60° C., in which the ORP water solution preferablyis stable for up to about 90 days, and more preferably for up to about180 days.

Stability also can be measured based on the concentration over time ofone or more species (or precursors thereof) present in solution duringthe shelf-life of the ORP water solution. Preferably, the concentrationsof one or more species, e.g., free chlorine, hypocholorous acid and oneor more additional superoxidized water species and are maintained atabout 70% or greater of their initial concentration for at least abouttwo months after preparation of the ORP water solution. More preferably,the concentration of one of more of these species is maintained at about80% or greater of their initial concentration for at least about twomonths after preparation of the ORP water solution. Still morepreferably, the concentration of one or more of such species ismaintained at about 90% or greater, and most preferably is maintained atabout 95% or greater, of their initial concentration for at least abouttwo months after preparation of the ORP water solution.

Stability also can be determined based on the reduction in the amount oforganisms present in a sample following exposure to the ORP watersolution. Measuring the reduction of organism concentration can be madeon the basis of any suitable organism including, e.g., bacteria, fungi,yeasts, or viruses. Suitable organisms can include, e.g., Escherichiacoli, Staphylococcus aureus, Candida albicans, and Bacillus athrophaeus(formerly B. subtilis).

The ORP water solution administered in accordance with the invention canfunction as a low-level disinfectant capable of a four log (10⁴)reduction in the concentration of live microorganisms, and also canfunction as a high-level disinfectant capable of a six log (10⁶)reduction in concentration of live microorganisms. Preferably, the ORPwater solution administered in accordance with the invention is capableof yielding at least about a four log (10⁴) reduction in total organismconcentration, following exposure for one minute when measured at leastabout two months after preparation of the solution. More preferably, theORP water solution is capable of a 10⁴-10⁶ reduction of organismconcentration when measured at least about six months after preparationof the solution. Still more preferably, the ORP water solution iscapable of a 10⁴-10⁶ reduction of organism concentration when measuredat least about one year after preparation of the ORP water solution, andmost preferably when measured more than about one year, e.g., at leastabout two years or at least about three years, after preparation of theORP water solution.

For instance, the ORP water solution administered in accordance with thepresent invention can be capable of at least about a five log (10⁵)reduction in the concentration of a sample of live microorganisms fromthe group consisting of Pseudomonas aeruginosa, Escherichia coli,Enterococcus hirae, Acinetobacter baumannii, Acinetobacter species,Bacteroides fragilis, Enterobacter aerogenes, Enterococcus faecalis,Vancomycin resistant-Enterococcus faecium (VRE, MDR), Haemophilusinfluenzae, Klebsiella oxytoca, Klebsiella pneumoniae, Micrococcusluteus, Proteus mirabilis, Serratia marcescens, Staphylococcus aureus,Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcushominis, Staphylococcus saprophyticus, Streptococcus pneumoniae,Streptococcus pyogenes, Candida albicans within thirty seconds ofexposure, when measured at least two months after preparation of the ORPwater solution (BioSciences Labs, Montana, US). Preferably, the ORPwater solution is capable of achieving a 10⁵ reduction of all theseorganisms when measured at least about six months after preparation, andmore preferably when measured at least about one year after preparation.

The invention also provides methods for killing bacteria in biofilms,e.g., Pseudomonas aeruginosa in biofilms. The invention further providesmethods for killing of Moraexlla catarrhalis and antibotic resistantbacteria, e.g., penicillin resistant Streptococcus. The methodsdisclosed herein can be used in accordance with the invention forkilling bacteria using ORP water solutions faster than with usingBacitracin.

In one embodiment, the ORP water solution administered in accordancewith the invention can reduce a sample of live microorganisms including,but not limited to, Escherichia coli, Pseudomonas aeruginosa,Staphylococcus aureus and Candida albicans, from an initialconcentration of between about 1×10⁶ and about 1×10⁸ organisms/ml to afinal concentration of about zero organisms/ml within about one minuteof exposure when measured at least about two months after preparation ofthe ORP water solution. This corresponds to from about a six log (10⁶)to about an eight log (10⁸) reduction in organism concentration.Preferably, the ORP water solution is capable of achieving a 10⁶-10⁸reduction of Escherichia coli, Pseudomonas aeruginosa, Staphylococcusaureus or Candida albicans organisms when measured at least about sixmonths after preparation, and more preferably when measured at leastabout one year after preparation.

Alternatively, the ORP water solution administered in accordance withthe present invention can produce about a six log (10⁶) reduction in theconcentration of a spore suspension of Bacillus athrophaeus sporeswithin about five minutes of exposure when measured at least about twomonths after preparation of the ORP water solution. Preferably, the ORPwater solution administered in accordance with the invention can achieveabout a 10⁶ reduction in the concentration of Bacillus athrophaeusspores when measured at least about six months after preparation, andmore preferably when measured at least about one year after preparation.

The ORP water solution administered in accordance with the inventionalso can produce about a four log (10⁴) reduction in the concentrationof a spore suspension of Bacillus athrophaeus spores within about thirty(30) seconds of exposure when measured at least about two months afterpreparation of the ORP water solution. Preferably, the ORP watersolution can achieve this reduction in the concentration of Bacillusathrophaeus spores when measured at least about six months afterpreparation, and more preferably when measured, at least about one yearafter preparation.

The ORP water solution administered in accordance with the inventionfurther can produce about a six log (10⁶) reduction in the concentrationof fungal spores, such as Aspergillis niger spores, within about five toabout ten minutes of exposure when measured at least about two monthsafter preparation of the ORP water solution. Preferably, the ORP watersolution can achieve a 10⁶ reduction in the concentration of fungalspores when measured at least about six months after preparation, andmore preferably when measured at least about one year after preparation.

The ORP water solution administered in accordance with the invention canbe acidic, neutral or basic, and generally can have a pH of from about 1to about 14. Within this pH range, the ORP water solution can be safelyapplied in suitable quantities, e.g., to surfaces without damaging thesurfaces or harming objects, such as human skin, that comes into contactwith the ORP water solution. Preferably, the pH of the ORP watersolution administered in accordance with the invention is from about 3to about 8. More preferably, the pH of the ORP water solution is fromabout 6.4 to about 7.8, and still more preferably, the pH is from about7.4 to about 7.6.

The ORP water solution administered in accordance with the invention canhave an oxidation-reduction potential of from about −1000 millivolts(mV) to about +1150 millivolts (mV). This potential is a measure of thetendency (i.e., the potential) of a solution to either accept ortransfer electrons that are sensed by a metal electrode and comparedwith a reference electrode in the same solution. This potential may bemeasured by standard techniques including, for example, measuring theelectrical potential in millivolts of the ORP water solution relative tostandard reference such as, e.g., a silver/silver chloride electrode.

The ORP water solution administered in accordance with the inventionpreferably has a potential of from about −400 mV to about +1300 mV. Morepreferably, the ORP water solution has a potential of from about 0 mV toabout +1250 mV, and still more preferably from about +500 mV to about+1250 mV. Even more preferably, the ORP water solution administered inaccordance with the present invention has a potential of from about +800mV to about +1100 mV, and most preferably from about +800 mV to about+1000 mV.

Various ionic and other species may be present in the ORP water solutionadministered in accordance with the invention. For example, the ORPwater solution may contain chlorine (e.g., free chlorine and boundchlorine), and dissolved oxygen and, optionally, ozone and peroxides(e.g., hydrogen peroxide). The presence of one or more of these speciesis believed to contribute to at least the disinfectant ability of theORP water solution to kill a variety of microorganisms, such as bacteriaand fungi, as well as viruses. Although not wishing to be bound by anyparticular theory, it is believed that or more of such species also maycontribute the anti-inflammatory efficacy of the ORP water solution.

Free chlorine typically includes, but is not limited to, hypochlorousacid (HClO), hypochlorite ions (ClO⁻), sodium hypochlorite (NaOCl), andprecursors thereof. The ratio of hypochlorous acid to hypochlorite ionis dependent upon pH. At a pH of 7.4, hypochlorous acid levels aretypically from about 25 ppm to about 75 ppm. Temperature also impactsthe ratio of the free chlorine component.

Bound chlorine typically includes chlorine in chemical combination with,e.g., ammonia or organic amines (e.g., chloramines). Bound chlorine ispreferably present in an amount of up to about 20 ppm.

One or more chlorine species, one or more additional superoxidized waterspecies (e.g., one or more additional oxidizing species such as, e.g.,oxygen) can be present in the ORP water solution administered inaccordance with the invention in any suitable amount. The levels ofthese components may be measured by any suitable method, includingmethods known in the art.

The total amount of free chlorine species is preferably from about 10ppm to about 400 ppm, more preferably from about 50 ppm to about 200ppm, and most preferably from about 50 ppm to about 80 ppm. The amountof hypochlorous acid is preferably from about 15 ppm to about 35 ppm.The amount of sodium hypochlorite is preferably in the range of fromabout 25 ppm to about 50 ppm. Optionally, Chlorine dioxide levels arepreferably less than about 5 ppm.

The chlorine content may be measured by methods known in the art, suchas the DPD colorimeter method (Lamotte Company, Chestertown, Md.) orother known methods such as, e.g., methods established by theEnvironmental Protection Agency. In the DPD colorimeter method, a yellowcolor is formed by the reaction of free chlorine withN,N-diethyl-p-phenylenediamine (DPD) and the intensity is measured witha calibrated calorimeter that provides the output in parts per million.Further addition of potassium iodide turns the solution a pink color toprovide the total chlorine value. The amount of bound chlorine presentis then determined by subtracting free chlorine from the total chlorine.

The total amount of oxidizing chemical species present in the ORP watersolution is preferably in the range of about 2 millimolar (mM), whichincludes the aforementioned chlorine species, oxygen species, andadditional species, including those, which can be difficult to measuresuch as, e.g., Cl⁻, ClO₃, Cl₂ ⁻, and ClO_(x).

In one embodiment, the ORP water solution administered in accordancewith the invention comprises one or more chlorine species and one ormore additional superoxidized water species (e.g., one or moreadditional oxidizing species such as, e.g., oxygen). Preferably, thechlorine species present is a free chlorine species. The free chlorinespecies can include one or more species selected from the groupconsisting of hypochlorous acid (HOCl), hypochlorite ions (OCl⁻), andsodium hypochlorite (NaOCl), chloride ion (Cl⁻), and optionally,chlorine dioxide (ClO₂), dissolved chlorine gas (Cl₂), precursorsthereof and mixtures thereof.

In one embodiment, the ORP water solution includes one or more chlorinespecies or one or more precursors thereof, and one or more additionalsuperoxidized water species or one or more precursors thereof, and,optionally, hydrogen peroxide, and is stable for at least about 24hours, preferably for at least about one week, more preferably for atabout least two months, and still more preferably for at least about sixmonths after its preparation. Even more preferably, such ORP watersolution is stable for at least about one year, and most preferably formore than about one year, e.g., at least about two years or at leastabout three years.

It is also preferred that the ORP water solution includes one or morechlorine species (e.g., hypocholorous acid and sodium hypochlorite) orone or more precursors thereof and one or one or more additionalsuperoxidized water species (e.g., one or more oxygen species, dissolvedoxygen) or one or more precursors thereof and has a pH of from about 6to about 8. More preferably from about 6.2 to about 7.8, and mostpreferably from about 7.4 to about 7.6. An exemplary ORP water solutionadministered in accordance with the present invention can comprise,e.g., from about 15 ppm to about 35 ppm hypochlorous acid, from about 25ppm to about 50 ppm sodium hypochlorite, from about 1 ppm to about 4 ppmof one or more additional superoxidized water species and a pH of fromabout 6.2 to about 7.8, and can be stable for at least about one week,e.g., at least about two months, at least about six months, at leastabout one year, or more than about one year, e.g., at least about twoyears or at least about three years.

While in no way limiting the present invention, it is believed that thecontrol of pH and other variables (e.g., salinity) can provide stableORP water solutions, which contain one or more chlorine species orprecursors thereof, such as, e.g., hypochlorous acid and hypochloriteions, and one or more additional superoxidized water species (e.g.,oxygen) or one or more precursors thereof.

The ORP water solutions administered in accordance with the inventionpreferably comprises one or more oxidized water species which can yieldfree radicals (such as, e.g., hydroxyl radicals) on exposure to iron.The ORP water can optionally include one or more chemical compoundsgenerated during the production thereof such as, e.g., sodium hydroxide(NaOH), chlorine dioxide (ClO₂), peroxides (e.g., hydrogen peroxide(H₂O₂), and ozone (O₃) although, it has been reported that sodiumhydroxide, chlorine dioxide, hydrogen peroxide, and ozone may react withhypocholrite resulting in their consumption and the production of otherchemical species.

The ORP water solution administered in accordance with the presentinvention can be produced by an oxidation-reduction process, e.g., by anelectrolytic process or redox reaction, in which electrical energy isused to produce one or more chemical changes in an aqueous solution.Exemplary processes for preparing suitable ORP water solutions aredescribed, e.g., in U.S. Patent Application Publication Nos. US2005/0139808 and US 2005/0142157 (hereby incorporated by reference).

In the electrolytic process, electrical energy is introduced into andtransported through water by the conduction of electrical charge fromone point to another in the form of an electrical current. In order forthe electrical current to arise and subsist there should be chargecarriers in the water, and there should be a force that makes thecarriers move. The charge carriers can be electrons, as in the case ofmetal and semiconductors, or they can be positive and negative ions inthe case of solutions. A reduction reaction occurs at the cathode whilean oxidation reaction occurs at the anode. At least some of thereductive and oxidative reactions that are believed to occur aredescribed in International Application WO 03/048421 A1.

As used herein, water produced at an anode is referred to as anode waterand water produced at a cathode is referred to as cathode water. Anodewater typically contains oxidized species produced from the electrolyticreaction while cathode water typically contains reduced species from thereaction. Anode water generally has a low pH, typically of from about 1to about 6.8. The anode water preferably contains chlorine in variousforms including, for example, chlorine gas, chloride ions, hydrochloricacid and/or hypochlorous acid, or one or more precursors thereof. Oxygenin various forms is also preferably present including, for example,oxygen gas, and possibly one or more species formed during production(e.g., peroxides, and/or ozone), or one or more precursors thereof.Cathode water generally has a high pH, typically from about 7.2 to about11. Cathode water can contain hydrogen gas, hydroxyl radicals, and/orsodium ions.

The ORP water solution administered in accordance with the invention caninclude a mixture of anode water (e.g., water produced in the anodechamber of an electrolytic cell) and cathode water (e.g., water producedin the cathode chamber of an electrolysis cell). Preferably, the ORPwater solution administered in accordance with the present inventioncontains cathode water, e.g., in an amount of from about 10% by volumeto about 90% by volume of the solution. More preferably, cathode wateris present in the ORP water solution in an amount of from about 10% byvolume to about 50% by volume, and still more preferably of from about20% by volume to about 40% by volume of the solution, e.g., from about20% by volume to about 30% by volume of the solution. Additionally,anode water can be present in the ORP water solution, e.g., in an amountof from about 50% by volume to about 90% by volume of the solution.Exemplary ORP water solutions can contain from about 10% by volume toabout 50% by volume of cathode water and from about 50% by volume toabout 90% by volume of anode water. The anode and cathode water can beproduced using the three-chambered electrolysis cell shown in FIG. 1.

The ORP water solution administered in accordance with the invention ispreferably produced using at least one electrolysis cell comprising ananode chamber, a cathode chamber and a salt solution chamber locatedbetween the anode and cathode chambers, wherein at least some of theanode and cathode water are combined such that the ORP water solutioncomprises anode water and cathode water. A diagram of an exemplary threechamber electrolysis cell that can be used in preparing an exemplary ORPwater solution is shown in FIG. 2.

The electrolysis cell 100 has an anode chamber 102, cathode chamber 104and salt solution chamber 106. The salt solution chamber is locatedbetween the anode chamber 102 and cathode chamber 104. The anode chamber102 has an inlet 108 and outlet 110 to permit the flow of water throughthe anode chamber 100. The cathode chamber 104 similarly has an inlet112 and outlet 114 to permit the flow of water through the cathodechamber 104. The salt solution chamber 106 has an inlet 116 and outlet118. The electrolysis cell 100 preferably includes a housing to hold allof the components together.

The anode chamber 102 is separated from the salt solution chamber by ananode electrode 120 and an anion ion exchange membrane 122. The anodeelectrode 120 may be positioned adjacent to the anode chamber 102 withthe membrane 122 located between the anode electrode 120 and the saltsolution chamber 106. Alternatively, the membrane 122 may be positionedadjacent to the anode chamber 102 with the anode electrode 120 locatedbetween the membrane 122 and the salt solution chamber 106.

The cathode chamber 104 is separated from the salt solution chamber by acathode electrode 124 and a cathode ion exchange membrane 126. Thecathode electrode 124 may be positioned adjacent to the cathode chamber104 with the membrane 126 located between the cathode electrode 124 andthe salt solution chamber 106. Alternatively, the membrane 126 may bepositioned adjacent to the cathode chamber 104 with the cathodeelectrode 124 located between the membrane 126 and the salt solutionchamber 106.

The electrodes preferably are constructed of metal to permit a voltagepotential to be applied between the anode chamber and cathode chamber.The metal electrodes are generally planar and have similar dimensionsand cross-sectional surface area to that of the ion exchange membranes.The electrodes are configured to expose a substantial portion of thesurface of the ion exchange members to the water in their respectiveanode chamber and cathode chamber. This permits the migration of ionicspecies between the salt solution chamber, anode chamber and cathodechamber. Preferably, the electrodes have a plurality of passages orapertures evenly spaced across the surface of the electrodes.

A source of electrical potential is connected to the anode electrode 120and cathode electrode 124 so as to induce an oxidation reaction in theanode chamber 102 and a reduction reaction in the cathode chamber 104.

The ion exchange membranes 122 and 126 used in the electrolysis cell 100may be constructed of any suitable material to permit the exchange ofions between the salt solution chamber 106 and the anode chamber 102such as, e.g., chloride ions (Cl⁻) and between the salt solution saltsolution chamber 106 and the cathode chamber 104 such as, e.g., sodiumions (Na⁺). The anode ion exchange membrane 122 and cathode ion exchangemembrane 126 may be made of the same or different material ofconstruction. Preferably, the anode ion exchange membrane comprises afluorinated polymer. Suitable fluorinated polymers include, for example,perfluorosulfonic acid polymers and copolymers such as perfluorosulfonicacid/PTFE copolymers and perfluorosulfonic acid/TFE copolymers. The ionexchange membrane may be constructed of a single layer of material ormultiple layers. Suitable ion exchange membrane polymers can include oneor more ion exchange membrane polymers marketed under the trademarkNafion®.

The source of the water for the anode chamber 102 and cathode chamber104 of the electrolysis cell 100 may be any suitable water supply. Thewater may be from a municipal water supply or alternatively pretreatedprior to use in the electrolysis cell. Preferably, the water ispretreated and is selected from the group consisting of softened water,purified water, distilled water, and deionized water. More preferably,the pretreated water source is ultrapure water obtained using reverseosmosis purification equipment.

The salt water solution for use in the salt water chamber 106 caninclude any aqueous salt solution that contains suitable ionic speciesto produce the ORP water solution. Preferably, the salt water solutionis an aqueous sodium chloride (NaCl) salt solution, also commonlyreferred to as a saline solution. Other suitable salt solutions caninclude other chloride salts such as potassium chloride, ammoniumchloride and magnesium chloride as well as other halogen salts such aspotassium and bromine salts. The salt solution can contain a mixture ofsalts.

The salt solution can have any suitable concentration. For example, thesalt solution can be saturated or concentrated. Preferably, the saltsolution is a saturated sodium chloride solution.

FIG. 2 illustrates what are believed to be various ionic speciesproduced in the three chambered electrolysis cell useful in connectionwith the invention. The three chambered electrolysis cell 200 includesan anode chamber 202, cathode chamber 204, and a salt solution chamber206. Upon application of a suitable electrical current to the anode 208and cathode 210, the ions present in the salt solution flowing throughthe salt solution chamber 206 migrate through the anode ion exchangemembrane 212 and cathode ion exchange membrane 214 into the waterflowing through the anode chamber 202 and cathode chamber 204,respectively.

Positive ions migrate from the salt solution 216 flowing through thesalt solution chamber 206 to the cathode water 218 flowing through thecathode chamber 204. Negative ions migrate from the salt solution 216flowing through the salt solution chamber 206 to the anode water 220flowing through the anode chamber 202.

Preferably, the salt solution 216 is aqueous sodium chloride (NaCl),which contains both sodium ions (Na⁺) and chloride ions (Cl⁻) ions.Positive Na⁺ ions migrate from the salt solution 216 to the cathodewater 218. Negative Cl⁻ ions migrate from the salt solution 216 to theanode water 220.

The sodium ions and chloride ions may undergo further reaction in theanode chamber 202 and cathode chamber 204. For example, chloride ionscan react with various oxygen ions and other species (e.g., oxygencontaining free radicals, O₂, O₃) present in the anode water 220 toproduce ClOn- and ClO⁻. Other reactions may also take place in the anodechamber 202 including the formation of oxygen free radicals, hydrogenions (H⁺), oxygen (e.g., as O₂), ozone (O₃), and peroxides. In thecathode chamber 204, hydrogen gas (H₂), sodium hydroxide (NaOH),hydroxide ions (OH⁻), and other radicals may be formed.

The apparatus for producing the ORP water solution also can beconstructed to include at least two three chambered electrolysis cells.Each of the electrolytic cells includes an anode chamber, cathodechamber, and salt solution chamber separating the anode and cathodechambers. The apparatus includes a mixing tank for collecting the anodewater produced by the electrolytic cells and a portion of the cathodewater produced by one or more of the electrolytic cells. Preferably, theapparatus further includes a salt recirculation system to permitrecycling of the salt solution supplied to the salt solution chambers ofthe electrolytic cells. A diagram of an exemplary process for producingan ORP water solution using two electrolysis cells is shown in FIG. 3.

The process 300 includes two three-chambered electrolytic cells,specifically a first electrolytic cell 302 and second electrolytic cell304. Water is transferred, pumped or otherwise dispensed from the watersource 305 to anode chamber 306 and cathode chamber 308 of the firstelectrolytic cell 302 and to anode chamber 310 and cathode chamber 312of the second electrolytic cell 304. Advantageously, this process canproduce from about 1 liter/minute to about 50 liters/minute of ORP watersolution. The production capacity may be increased by using additionalelectrolytic cells. For example, three, four, five, six, seven, eight,nine, ten or more three-chambered electrolytic cells may be used toincrease the output of the ORP water solution administered in accordancewith the invention.

The anode water produced in the anode chamber 306 and anode chamber 310are collected in the mixing tank 314. A portion of the cathode waterproduced in the cathode chamber 308 and cathode chamber 312 is collectedin mixing tank 314 and combined with the anode water. The remainingportion of cathode water produced in the process is discarded. Thecathode water may optionally be subjected to gas separator 316 and/orgas separator 318 prior to addition to the mixing tank 314. The gasseparators remove gases such as hydrogen gas that are formed in cathodewater during the production process.

The mixing tank 314 may optionally be connected to a recirculation pump315 to permit homogenous mixing of the anode water and portion ofcathode water from electrolysis cells 302 and 304. Further, the mixingtank 314 may optionally include suitable devices for monitoring thelevel and pH of the ORP water solution. The ORP water solution may betransferred from the mixing tank 314 via pump 317 for application indisinfection or sterilization at or near the location of the mixingtank. Alternatively, the ORP water solution may be dispensed into one ormore suitable containers for shipment to a remote site (e.g., warehouse,hospital, etc.).

The process 300 further includes a salt solution recirculation system toprovide the salt solution to salt solution chamber 322 of the firstelectrolytic cell 302 and the salt solution chamber 324 of the secondelectrolytic cell 304. The salt solution is prepared in the salt tank320. The salt is transferred via pump 321 to the salt solution chambers322 and 324. Preferably, the salt solution flows in series through saltsolution chamber 322 first followed by salt solution chamber 324.Alternatively, the salt solution may be pumped to both salt solutionchambers simultaneously.

Before returning to the salt tank 320, the salt solution may flowthrough a heat exchanger 326 in the mixing tank 314 to control thetemperature of the ORP water solution as needed.

The ions present in the salt solution are depleted over time in thefirst electrolytic cell 302 and second electrolytic cell 304. Anadditional source of ions periodically can be added to the mixing tank320 to replace the ions that are transferred to the anode water andcathode water. The additional source of ions may be used, e.g., tomaintain a constant pH of the salt solution, which can to drop (i.e.,become acidic) over time. The source of additional ions may be anysuitable compound including, for example, salts such as, e.g., sodiumchloride. Preferably, sodium hydroxide is added to the mixing tank 320to replace the sodium ions (Na⁻) that are transferred to the anode waterand cathode water.

Following its preparation, the ORP water solution can be transferred toone or more suitable containers, e.g., a sealed container fordistribution and sale to end users such as, e.g., health care facilitiesincluding, e.g., hospitals, nursing homes, doctor offices, outpatientsurgical centers, dental offices, and the like. Suitable containers caninclude, e.g., a sealed container that maintains the sterility andstability of the ORP water solution held by the container. The containercan be constructed of any material that is compatible with the ORP watersolution. Preferably, the container is generally non-reactive with oneor more ions or other species present in the ORP water solution.

Preferably, the container is constructed of plastic or glass. Theplastic can be rigid so that the container is capable of being stored ona shelf Alternatively, the container can be flexible, e.g., a containermade of flexible plastic such as, e.g., a flexible bag.

Suitable plastics can include, e.g., polypropylene, polyesterterephthalate (PET), polyolefin, cycloolefin, polycarbonate, ABS resin,polyethylene, polyvinyl chloride, and mixtures thereof. Preferably, thecontainer comprises one or more polyethylenes selected from the groupconsisting of high-density polyethylene (HDPE), low-density polyethylene(LDPE), and linear low-density polyethylene (LLDPE). Most preferably,the container is constructed of high density polyethylene.

The container preferably has an opening to permit dispensing of the ORPwater solution. The container opening can be sealed in any suitablemanner. For example, the container can be sealed with a twist-off cap orstopper. Optionally, the opening can be further sealed with a foillayer.

The headspace gas of the sealed container can be air or any othersuitable gas, which preferably does not react with one or more speciesin the ORP water solution. Suitable headspace gases can include, e.g.,nitrogen, oxygen, and mixtures thereof.

The ORP water solution administered in accordance with the inventionalso can be used for the prevention or treatment of an infection, e.g.,by one or more infectious pathogens such as, for example, infectiousmicroorganisms. Such microorganisms can include, for example, viruses,bacteria, and fungi. The viruses can include, e.g., one or more virusesselected from the group consisting of adenoviruses, herpes viruses,coxsackie viruses, HIV, rhinoviruses, cornaviruses, and flu viruses. Thebacteria can include, e.g., one or more bacteria selected from the groupconsisting of Escherichia coli, Pseudomonas aeruginosa, Staphylococcusaureus, and Mycobaterium tuberculosis. The fungi can include, e.g., oneor more fungi selected from the group consisting of Candida albicans,Bacillus subtilis and Bacillus athrophaeus.

The ORP water solution administered in accordance with the inventionalso can be effective against adenovirus. Preferably, the ORP watersolution administered in accordance with the invention preferablyachieves a log-10 reduction in the adenoviral load of greater than about2, more preferably greater than about 2.5, and still more preferablygreater than about 3, after exposure to the ORP water solution for about20 minutes, more preferably after exposure for about 15 minutes, andstill more preferably after exposure for about 10 minutes. The ORP watersolution administered in accordance with the invention also can beeffective for reducing the viral load of HIV-1, preferably by a logreduction factor greater than about 2, more preferably by a logreduction factor of greater than about 2.5, and still more preferably bya log reduction factor of greater than about 3 after exposure to the ORPwater solution for about five minutes.

In accordance with the method of the present invention, administeringthe ORP water solution for the prevention or treatment of infection alsocan serve to prevent or treat inflammation associated with the infection(or the affected tissues) as described herein.

The ORP water solution administered in accordance with the inventionalso can be used for treating impaired or damaged tissue, e.g., bycontacting one or more impaired or damaged tissues with atherapeutically effective amount of the ORP water solution. Any suitablemethod can be used for contacting the impaired or damaged tissue, so asto treat the impaired or damaged tissue. For example, the impaired ordamaged tissue can be treated by irrigating the tissue with the ORPwater solution, so as to contact the impaired or damaged tissue with atherapeutically effective amount of the ORP water solution. The ORPwater solution can be administered as a steam or a spray, or byaerosolization, nebulization or atomization, as described herein, so asto contact the impaired or damaged tissue with a therapeuticallyeffective amount of the ORP water solution.

The ORP water solution administered in accordance with the invention canbe used for treating tissues, which have been impaired or damaged, e.g.,by surgery. For instance, the ORP water solution can be used fortreating tissues, which have been impaired or damaged by an incision. Inaddition, the ORP water solution can be used for treating tissues, whichhave been impaired or damaged by oral surgery, graft surgery, implantsurgery, transplant surgery, cauterization, amputation, radiation,chemotherapy, and combinations thereof. The oral surgery can include,for example, dental surgery such as, e.g., root canal surgery, toothextraction, gum surgery, and the like.

The ORP water solution administered in accordance with the invention canbe used for treating tissues, which have been impaired or damaged by oneor more burns, cuts, abrasions, scrapes, rashes, ulcers, puncturewounds, combinations thereof, and the like, which are not necessarilycaused by surgery. The ORP water solution administered in accordancewith the invention can be used for treating impaired or damaged tissue,which is infected, or tissue impaired or damaged due to infection. Suchinfection can be caused by one or more infectious pathogens, such as,e.g., one or more microorganisms selected from the group consisting ofviruses, bacteria, and fungi, as described herein.

In accordance with the present invention, administering the ORP watersolution for treating impaired or damaged tissue also can serve toprevent or treat inflammation associated with the impairment or damage(or with the impaired or damaged tissue).

The ORP water solution administered in accordance with the inventionalso can be used as a disinfectant to eradicate microorganisms,including bacteria, viruses and spores, in a variety of settings, e.g.,in the healthcare and medical device fields, to disinfect surfaces andmedical equipment, and also can be applied in wound care, medical devicesterilization, food sterilization, hospitals, consumer households andanti-bioterrorism. The ORP water solution can be used for disinfecting asurface, e.g., by contacting the surface with an anti-infective amountof the ORP water solution. The surface can be contacted using anysuitable method. For example, the surface can be contacted by irrigatingthe surface with the ORP water solution, so as to disinfect the surface.Additionally, the surface can be contacted by applying the ORP watersolution to the surface as a steam or a spray, or by aerosolization,nebulization or atomization, as described herein, so as to disinfect thesurface. Further, the ORP water solution can be applied to the surfacewith a cleaning wipe, as described herein. By disinfecting a surface,the surface may be cleansed of infectious microorganisms. Alternatively(or additionally), the ORP water solution administered in accordancewith the present invention can be applied to the surface to provide abarrier to infection, to thereby disinfect the surface.

The surface(s) can include one or more biological surfaces, one or moreinanimate surfaces, and combinations thereof. Biological surfaces caninclude, for example, tissues within one or more body cavities such as,for example, the oral cavity, the sinus cavity, the cranial cavity, theabdominal cavity, and the thoracic cavity. Tissues within the oralcavity include, e.g., mouth tissue, gum tissue, tongue tissue, andthroat tissue. The biological tissue also can include muscle tissue,bone tissue, organ tissue, mucosal tissue, vascular tissue, neurologicaltissue, and combinations thereof. Biological surfaces also include anyother cultured tissue in vitro, such as primary and established celllines, stem cells of any nature, xenotransplants, tissue substitutes(e.g. made of collagen or any other organic material in addition or notof cellular elements), any other tissue-engineered substitutes andcombinations thereof.

Inanimate surfaces include, for example, surgically implantable devices,prosthetic devices, and medical devices. In accordance with the methodof the present invention, the surfaces of internal organs, viscera,muscle, and the like, which may be exposed during surgery, can bedisinfected, e.g., to maintain sterility of the surgical environment. Inaccordance with the present invention, administering the ORP watersolution for disinfecting a surface also can serve to treat or preventinflammation affecting one or more biological tissues associated withsuch surfaces.

The ORP water solution may also be applied to humans and/or animals totreat various conditions, including inflammation, hypersensitivity, andassociated systemic effects associated with one or more of thefollowing: surgical/open wound cleansing agent; skin pathogendisinfection (e.g., for bacteria, mycoplasmas, virus, fungi, prions);battle wound disinfection; wound healing promotion; burn healingpromotion; treatment of stomach ulcers; wound irrigation; skin fungi;psoriasis; athlete's foot; pinkeye and other eye infections; earinfections (e.g., swimmer's ear); lung/nasal/sinus infections; and othermedical applications on or in the human or animal body, as well asenvironmental remediation. The use of ORP water solutions as a tissuecell growth promoter is further described in U.S. Patent ApplicationPublication 2002/0160053 (hereby incorporated by reference).

The ORP water solution may be used as a disinfectant, sterilizationagent, decontaminant, antiseptic and/or cleanser. The ORP water solutionadministered in accordance with the invention is suitable for use in thefollowing representative applications: medical, dental and/or veterinaryequipment and devices; food industry (e.g., hard surfaces, fruits,vegetables, meats); hospitals/health care facilities (e.g., hardsurfaces); cosmetic industry (e.g., skin cleaner); households (e.g.,floors, counters, hard surfaces); electronics industry (e.g., cleaningcircuitry, hard drives); and bio-terrorism (e.g., anthrax, infectiousmicrobes).

Organisms that can be controlled, reduced, killed or eradicated bytreatment with the ORP water solution include, but are not limited to,bacteria, fungi, yeasts, and viruses. Susceptible bacteria include, butare not limited to, Escherichia coli, Staphylococcus aureus, Bacillusathrophaeus, Streptococcus pyogenes, Salmonella choleraesuis,Pseudomonas aeruginosa, Shingella dysenteriae, and other susceptiblebacteria. Fungi and yeasts that may be treated with the ORP watersolution include, for example, Candida albicans and Trichophytonmentagrophytes. The ORP water solution may also be applied to virusesincluding, for example, adenovirus, human immunodeficiency virus (HIV),rhinovirus, influenza (e.g., influenza A), hepatitis (e.g., hepatitisA), coronavirus (responsible for Severe Acute Respiratory Syndrome(SARS)), rotavirus, respiratory syncytial virus, herpes simplex virus,varicella zoster virus, rubella virus, and other susceptible viruses.

The ORP water solution may be applied to disinfect and sterilize in anysuitable manner. For example, to disinfect and sterilize medical ordental equipment, the equipment can be maintained in contact with theORP water solution for a sufficient period of time to reduce the levelof organisms present on the equipment to a desired level. Alternatively,the ORP water solution can be applied to medical or dental equipment byimmersing the equipment in a container with or without the applicationof enhancing physical procedures, e.g. ultrasound, shakers, heaters, andthe like.

For disinfection and sterilization of hard surfaces, the ORP watersolution can be applied to the hard surface directly from a container inwhich the ORP water solution is stored. For example, the ORP watersolution can be poured, sprayed or otherwise directly applied to thehard surface. The ORP water solution can then be distributed over thehard surface using a suitable substrate such as, for example, cloth,fabric or paper towel. In hospital applications, the substrate ispreferably sterile. Alternatively, the ORP water solution can first beapplied to a substrate such as cloth, fabric or paper towel. The wettedsubstrate can then be contacted with the hard surface. Alternatively,the ORP water solution can be applied to hard surfaces by dispersing thesolution into the air as described herein. The ORP water solution can beapplied in a similar manner to humans and animals.

The ORP water solution also can be applied with a cleaning wipecomprising a water insoluble substrate and the ORP water solution asdescribed herein, wherein the ORP water solution is dispensed onto thesubstrate. The ORP water solution can be impregnated, coated, covered orotherwise applied to the substrate. Preferably, the substrate ispretreated with the ORP water solution before distribution of thecleaning wipes to end users.

The substrate for the cleaning wipe can be any suitable water-insolubleabsorbent or adsorbent material. A wide variety of materials can be usedas the substrate. It should have sufficient wet strength, abrasivity,loft and porosity. Further, the substrate should not adversely impactthe stability of the ORP water solution. Examples include non wovensubstrates, woven substrates, hydroentangled substrates and sponges.

The substrate can have one or more layers. Each layer can have the sameor different textures and abrasiveness. Differing textures can resultfrom the use of different combinations of materials or from the use ofdifferent manufacturing processes or a combination thereof. Thesubstrate should not dissolve or break apart in water. The substrate canthereby provide a vehicle for delivering the ORP water solution to thesurface to be treated.

The substrate can be a single nonwoven sheet or multiple nonwovensheets. The nonwoven sheet can be made of wood pulp, synthetic fibers,natural fibers, and blends thereof. Suitable synthetic fibers for use inthe substrate can include, without limitation, polyester, rayon, nylon,polypropylene, polyethylene, other cellulose polymers, and mixtures ofsuch fibers. The nonwovens can include nonwoven fibrous sheet materialswhich include meltblown, coform, air-laid, spun bond, wet laid,bonded-carded web materials, hydroentangled (also known as spunlaced)materials, and combinations thereof. These materials can comprisesynthetic or natural fibers or combinations thereof. A binder canoptionally be present in the substrate.

Examples of suitable nonwoven, water insoluble substrates include 100%cellulose Wadding Grade 1804 from Little Rapids Corporation, 100%polypropylene needlepunch material NB 701-2.8-W/R from AmericanNon-wovens Corporation, a blend of cellulosic and syntheticfibres-Hydraspun 8579 from Ahlstrom Fibre Composites, and 70%Viscose/30% PES Code 9881 from PGI Nonwovens Polymer Corp. Additionalexamples of nonwoven substrates suitable for use in the cleaning wipesare described in U.S. Pat. Nos. 4,781,974, 4,615,937, 4,666,621, and5,908,707, and International Patent Application Publications WO98/03713, WO 97/40814, and WO 96/14835 (all herby incorporated byreference.).

The substrate also can be made of woven materials, such as cottonfibers, cotton/nylon blends, or other textiles. Regenerated cellulose,polyurethane foams, and the like, which are used in making sponges, alsocan be suitable for use.

The liquid loading capacity of the substrate should be at least about50%-1000% of the dry weight thereof, most preferably at least about200%-800%. This is expressed as loading 1/2 to 10 times the weight ofthe substrate. The weight of the substrate varies without limitationfrom about 0.01 to about 1,000 grams per square meter, most preferably25 to 120 grams/m² (referred to as “basis weight”) and typically isproduced as a sheet or web which is cut, die-cut, or otherwise sizedinto the appropriate shape and size. The cleaning wipes will preferablyhave a certain wet tensile strength which is without limitation about 25to about 250 Newtons/m, more preferably about 75-170 Newtons/m.

The ORP water solution can be dispensed, impregnated, coated, covered orotherwise applied to the substrate by any suitable method. For example,individual portions of substrate can be treated with a discrete amountof the ORP water solution. Preferably, a mass treatment of a continuousweb of substrate material with the ORP water solution is carried out.The entire web of substrate material can be soaked in the ORP watersolution. Alternatively, as the substrate web is spooled, or even duringcreation of a nonwoven substrate, the ORP water solution can be sprayedor metered onto the web. A stack of individually cut and sized portionsof substrate can be impregnated or coated with the ORP water solution inits container by the manufacturer.

The cleaning wipes optionally can contain additional components toimprove the properties of the wipes. For example, the cleaning wipes canfurther comprise polymers, surfactants, polysaccharides,polycarboxylates, polyvinyl alcohols, solvents, chelating agents,buffers, thickeners, dyes, colorants, fragrances, and mixtures thereofto improve the properties of the wipes. These optional components shouldnot adversely impact the stability of the ORP water solution. Examplesof various components that may optionally be included in the cleaningwipes are described in U.S. Pat. Nos. 6,340,663, 6,649,584 and 6,624,135(hereby incorporated by reference).

The cleaning wipes can be individually sealed with a heat-sealable orglueable thermoplastic overwrap (such as polyethylene, Mylar, and thelike). The wipes can also be packaged as numerous, individual sheets formore economical dispensing. The cleaning wipes can be prepared by firstplacing multiple sheets of the substrate in a dispenser and thencontacting the substrate sheets with the ORP water solution administeredin accordance with the invention. Alternatively, the cleaning wipes canbe formed as a continuous web by applying the ORP water solution to thesubstrate during the manufacturing process and then loading the wettedsubstrate into a dispenser.

The dispenser includes, but is not limited to, a canister with aclosure, or a tub with closure. The closure on the dispenser is to sealthe moist wipes from the external environment and to prevent prematurevolatilization of the liquid ingredients.

The dispenser can be made of any suitable material that is compatiblewith both the substrate and the ORP water solution. For example, thedispenser can be made of plastic, such as high density polyethylene,polypropylene, polycarbonate, polyethylene terephthalate (PET),polyvinyl chloride (PVC), or other rigid plastics.

The continuous web of wipes can be threaded through a thin opening inthe top of the dispenser, most preferably, through the closure. A meansof sizing the desired length or size of the wipe from the web can thenbe desirable. A knife blade, serrated edge, or other means of cuttingthe web to desired size can be provided on the top of the dispenser, fornon-limiting example, with the thin opening actually doubling in duty asa cutting edge. Alternatively, the continuous web of wipes can bescored, folded, segmented, perforated or partially cut into uniform ornon-uniform sizes or lengths, which would then obviate the need for asharp cutting edge. Further, the wipes can be interleaved, so that theremoval of one wipe advances the next.

The ORP water solution alternatively can be dispersed into theenvironment through a gaseous medium, such as air. The ORP watersolution can be dispersed into the air by any suitable means. Forexample, the ORP water solution can be formed into droplets of anysuitable size and dispersed into a room.

For small scale applications, the ORP water solution can be dispensedthrough a spray bottle that includes a standpipe and pump.Alternatively, the ORP water solution can be packaged in aerosolcontainers. Aerosol containers can include the product to be dispensed,propellant, container, and valve. The valve can include both an actuatorand dip tube. The contents of the container can be dispensed by pressingdown on the actuator. The various components of the aerosol containershould be compatible with the ORP water solution. Suitable propellantscan include a liquefied halocarbon, hydrocarbon, orhalocarbon-hydrocarbon blend, or a compressed gas such as carbondioxide, nitrogen, or nitrous oxide. Aerosol systems preferably yielddroplets that range in size from about 0.15 μm to about 5 μm.

Applications can also be conducted by using various hydrosurgeryequipments for debriding and cleaning (e.g VersaJet devices sold in theUnited States by Smith and Nephew, Debritom in Europe by Medaxis, JetOxin the United States and Europe by DeRoyal or PulsaVac in Italy),irrigation systems with negative pressure (e.g., VAC Instill), and thelike.

Optionally, several adjuvant therapies can also be utilized inaccordance with the invention including bioengineered skin (Apligraf,Organogenesis, Inc., Canton), acellular skin substitutes (Oasis WoundMatrix, Healthpoint), ultrasonic application of ORP water solutions, andlocal oxygen replacement or hyperbaric oxygen treatment (such as, e.g.,hyperbaric boots, the Vent-Ox System).

For some applications, the ORP water solution optionally can contain ableaching agent. The bleaching agent can include, e.g., any suitablecompound that lightens or whitens a substrate. The ORP water solutioncontaining a bleaching agent can be used in home laundering to disinfectand sterilize bacteria and germs as well as brighten clothing. Suitablebleaching agents include, but are not limited to, chlorine-containingbleaching agents and peroxide-containing bleaching agents. Mixtures ofbleaching agents also can be added to the ORP water solution.Preferably, the bleaching agent is added in the form of an aqueoussolution to the ORP water solution.

Suitable chlorine-containing bleaching agents can include, e.g.,chlorine, hypochlorites, N-chloro compounds, and chlorine dioxide.Preferably, the chlorine-containing bleaching agent added to the ORPwater solution is sodium hypochlorite or hypochlorous acid. Othersuitable chlorine-containing bleaching agents include, e.g., chlorine,calcium hypochlorite, bleach liquor (e.g., aqueous solution of calciumhypochlorite and calcium chloride), bleaching powder (e.g., mixture ofcalcium hypochlorite, calcium hydroxide, calcium chloride, and hydratesthereof), dibasic magnesium hypochlorite, lithium hypochlorite,chlorinated trisodium phosphate and mixtures thereof.

The addition of a bleaching agent to the ORP water solution can becarried out in any suitable manner. Preferably, an aqueous solutioncontaining the bleaching agent is first prepared. The aqueous solutioncontaining the bleaching agent can be prepared using household bleach(e.g., Clorox® bleach) or other suitable source of chlorine-containingbleaching agent or other bleaching agent. The bleaching agent solutioncan then be combined with the ORP water solution.

The bleaching agent can be added to the ORP water solution in anysuitable amount. Preferably, the ORP water solution containing ableaching agent is non-irritating to human or animal skin. Preferably,the total chloride ion content of the ORP water solution containing achlorine-containing bleaching agent is from about 1000 ppm to about 5000ppm, and preferably from about 1000 ppm to about 3000 ppm. The pH of theORP water solution containing a chlorine-containing bleaching agent ispreferably from about 8 to about 10, and the oxidative-reductivepotential is preferably from about +700 mV to about +800 mV.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting in its scope.

EXAMPLES 1-3

These examples demonstrate the unique features of the ORP water solutionused in accordance with the invention. The samples of the ORP watersolution in Examples 1-3 were analyzed in accordance with the methodsdescribed herein to determine the physical properties and levels ofionic and other chemical species present in each sample. The resultsobtained for chlorine dioxide, ozone and hydrogen peroxide are based onstandard tests used to measure such species but may be indicative ofdifferent species, which can also generate positive test results.Further, it has been reported that chlorine dioxide, ozone and hydrogenperoxide react with hypocholrite resulting in their consumption and theproduction of other compounds (e.g., HCl and O₂.) The pH,oxidative-reductive potential (ORP) and ionic species present are setforth in Table 1 for each sample of the ORP water solution.

TABLE 1 Physical characteristics and ion species present for the ORPwater solution samples EXAMPLE 1 EXAMPLE 2 EXAMPLE 3 pH 7.45 7.44 7.45ORP (mV) +879 +881 +874 Total Cl⁻ (ppm) 110 110 120 Bound Cl⁻ (ppm) 5 66

The ORP water solution has suitable physical characteristics for use in,e.g., disinfection, sterilization, cleaning, and/or the preventionand/or treatment of inflammation, sinusitis, peritonitis, or infection.

EXAMPLES 4-10

These examples demonstrate the addition of a bleaching agent to the ORPwater solution according to the invention in various amounts. Inparticular, these examples demonstrate the antimicrobial activity andfabric bleaching ability of the compositions.

A 10% Clorox® bleach solution was prepared using distilled water. Thefollowing solutions were then prepared using the 10% bleach solution:80% ORP water solution/20% bleach (Example 4); 60% ORP watersolution/40% bleach (Example 5); 40% ORP water solution/60% bleach(Example 6); 20% ORP water solution/80% bleach (Example 7); and 0% ORPwater solution/100% bleach (Example 8). Two control solutions were alsoused for comparison including 100% ORP water solution/0% bleach (Example9) and an ORP water solution with 0.01% Tween 20 detergent (Example 10).The physical characteristics of these samples were determined,specifically pH, oxidative-reductive potential (ORP), total chlorine(Cl⁻) content, hypochlorous acid (HClO⁻) content, and were tested forchlorine dioxide content and peroxide content, the results are set forthin Table 2.

TABLE 2 Physical characteristics of ORP water solution/bleachcompositions Total Cl⁻ HC1O⁻ pH ORP (ppm) (ppm) Ex. 4 8.92 +789 1248 62Ex. 5 9.20 +782 2610 104 Ex. 6 9.69 +743 4006 80 Ex. 7 9.86 +730 4800 48Ex. 8 9.80 +737 5000 50 Ex. 9 7.06 +901 64 32  Ex. 10 6.86 +914 51 26

The large bolus of chlorine ions added as part of the bleaching agentprevented the accurate measurement of the chlorine dioxide and peroxidelevels as indicated with the n.d. designations. Also, the resultsobtained for chlorine dioxide and peroxide are based on standard testsused to measure such species but may be indicative of different species,which can also generate positive test results. Further, it has beenreported that chlorine dioxide, ozone and hydrogen peroxide react withhypocholrite resulting in their consumption and the production of othercompounds (e.g., HCl and O₂.) As these examples demonstrate, thehypochlorous acid levels of the ORP water solution with and without theaddition of a bleaching agent are similar.

The samples of Examples 4-10 were subjected to a high spore count testusing Bacillus subtilis var. niger spores (ATCC #9372 obtained from SPSMedical of Rush, N.Y.). Spore suspensions were concentrated (byevaporation in a sterile hood) to 4×10⁶ spores per 100 microliters. A100 microliter sample of the spore suspension were mixed with 900microliters of each of the samples in Examples 4-10. The samples wereincubated at room temperature for periods of 1 to 5 minutes as set forthin Table 3. At the indicated times, 100 microliters of the incubatedsamples were plated onto individual TSA plates and incubated for 24hours at 35° C.±2° C., after which the number of resulting colonies oneach plate was determined. The control plates demonstrated that thestarting spore concentrations were >1×10⁶ spores/100 microliters. Theconcentration of Bacillus spores for the various samples at the variousincubation times (as the average of two determinations) is set forth inTable 3.

TABLE 3 Bacillus spore concentrations 1 minute 2 minutes 3 minutes 4minutes 5 minutes Ex. 4 >>1000 411 1 0 2 Ex. 5 >>1000 1000 1 0 0 Ex.6 >>1000 >>1000 >1000 22 0 Ex. 7 >>1000 >>1000 >1000 15 0 Ex.8 >>1000 >>1000 >1000 3 1 Ex. 9 >>1000 74 0 0 0  Ex 10 >>1000 239 3 0 0

As these results demonstrate, as the concentration of bleach (as 10%aqueous bleach solution) increases, the amount of Bacillus spores killedis reduced for the samples incubated for 2-3 minutes. However, forsamples incubated for 5 minutes, the bleach concentration does notimpact Bacillus spore kill. Further, the results demonstrate that theaddition of 0.01% detergent to the ORP water solution does not reducespore kill.

The samples of Examples 4-10 were subjected to a fabric bleaching test.The fabric upon which the samples were tested was a 100% rayonchildren's t-shirt with dark blue dye patches. Two inch square pieces ofdyed fabric were placed into 50 mL plastic tubes. Each fabric piece wascovered by a sample of the solution in Examples 4-10. The elapsed timeuntil complete bleaching was obtained, as determined by the whitening ofthe fabric, is set forth in Table 4.

TABLE 4 Time until complete bleaching of fabric sample Example Time Ex.4 39 minutes Ex. 5 23 minutes Ex. 6 18 minutes Ex. 7 19 minutes Ex. 8 10minutes Ex. 9 >6 hours  Ex. 10 >6 hours

As demonstrated by these examples, as the concentration of the ORP watersolution increases in the composition, the time until complete bleachingis achieved increases.

EXAMPLE 11

The purpose of this study was to assess the safety of the test anexemplary ORP water solution, Microcyn, when administered as drops intothe nasal cavity of rabbits. Thirty-three rabbits were randomly assignedto two groups, Groups I and II. Group I (18 animals) served as thecontrol group and Group II (15 animals) was dosed with the test article.On Day −1 or Day 0, body weights were recorded and blood samples were,collected for analysis of selected parameters. On Day 0, 500 μL ofsterile saline was administered to the Group I animals and 500 μL of thetest article (at a 50% concentration) was administered to Group nannuals. Both the control and the test articles were administered twicedaily as drops into the right nostril. The animals were dosed in thesame manner on Days 1-6. Animals were observed daily for signs ofpharmacologic and/or toxicologic effects with special attention paid tothe nose. Body weights were recorded weekly through study termination.On Day 7, one-third of the animals from each group were selected forblood collection, sacrifice and necropsy. The remaining animalscontinued to be dosed through Day 14, when half of the animals from eachgroup were selected for blood collection, sacrifice and necropsy. On Day21, after a 7-day recovery period), the remaining animals had bloodcollected and were sacrificed and necropsied. Samples of the nasalmucosa from both nostrils were collected from each animal forhistopathological analysis.

The necropsy consisted of gross observations of the respiratory tract.The entire nasal passage and associated bone were taken and fixed inbuffered formalin. Samples of any visible abnormalities in therespiratory tract were also collected for histopathology. Three biopsysamples (anterior, middle and posterior nasal cavity) per nostril(treated right and untreated left) were examined. The microscopichistopathology of the nasal mucosa included: integrity of epithelium,presence or loss of epithelial cilia, inflammatory cell infiltration,edema, presence of goblet cells, hyperplasia of glands, changes innumber or characteristics of blood vessels and any other changes orobservations.

The results (in-life observations including nasal observations, bodyweights, blood analysis, gross necropsy and histopathology results) fromthe test group were compared to the control group. The test group wasnot significantly different from animals treated with saline in terms ofmild irritation.

EXAMPLE 12

This example illustrates the lack of toxicity from the use of anexemplary ORP water solution.

The characterization of local and systemic toxicity from topicallyapplied Microcyn 60 to a deep wound was evaluated in rats. Noabnormalities, significant differences in the parameters of the bloodchemistry or hematic cytology were observed, nor anomalies in theautopsies. The skin irritation gradings and the histopathology of thewounds and the tissues around the place of application did not revealany difference between the wounds treated with Microcyn 60 and those ofthe control group treated with saline solution.

The systemic toxicity of Microcyn 60 was also evaluated by means of anintraperitoneal injection in mice. For this, five mice were injectedwith a single dose (50 mL/kg) of Microcyn 60 by the intraperitonealroute. In the same way, five control mice were injected with a singledose (50 mL/kg) of saline solution (sodium chloride at 0.9%). In thisinvestigation, neither mortality nor any evidence of systemic toxicitywas observed in any of the animals that received the singleintraperitoneal dose of Microcyn 60, indicating that the LD₅₀ is above50 mL/kg.

Microcyn 60 was administered by the oral route to rats to allow itsabsorption and to characterize any inherent toxic effect of the product.In this study, a single dose (4.98 mL/kg) was administered by esophagealtube to three albino rats of the Sprague-Dawley strain. There was nomortality, nor were there clinical signs or abnormalities in theautopsies of any of the animals exposed to the single oral dose ofMicrocyn 60.

The potential of topically applied Microcyn 60 for ocular irritation wasalso evaluated in rabbits. Ocular irritation was not observed nor anyother clinical sign in any animal exposed to Microcyn 60 by topicaladministration through the ocular route.

Microcyn 60 was applied by the inhalatory route to rats to determinepotential acute toxicity by inhalation. All the animals showed a veryslight or slight reduction in activity and piloerection after theexposure, but they were all asymptomatic on the following day. Mortalityor abnormalities were not observed at autopsy of the animals exposed toMicrocyn 60 by inhalation.

Evaluation of the potential for sensitization of the skin with Microcyn60 was carried out in guinea pigs using a modified occlusion patchmethod (Buehler). Irritation was not observed in the animals of thecontrol group after a simple treatment challenge, nor in the animalsevaluated (treated by induction) after challenge with the treatment.These studies demonstrate that Microcyn 60 does not provoke asensitizing reaction.

Thus, when it has been applied to the intact skin, deep open dermalwounds, in the conjunctival sac, by oral and inhalation routes or bymeans of intraperitoneal injection, Microcyn 60 has not shown adverseeffects related to the product. There is also experience in havingtreated more than thousands of patients with wounds of very diversenature in the skin and mucosae, with excellent antiseptic and cosmeticresults. Accordingly, topically applied Microcyn 60 should be effectiveand well-tolerated in this clinical trial.

Microcyn 60 is packaged in transparent 240 mL PET sealed bottles. Thisproduct is stored at ambient temperature and remains stable for up to 2years in such bottles. From its profile of high biological safety,Microcyn 60 can be safely disposed of, e.g., emptied into the sinkwithout risk of contamination or corrosion.

EXAMPLE 13

This example illustrates a clinical study, which can be used todetermine the effectiveness of an exemplary ORP water solution fortreating pharyngitis.

Multiple microbial trials have been run with Microcyn 60, both in theUnited States and in Mexico. Eradication of more than 90% of thebacteria occurs in the first few seconds of exposure. The antibacterialand antimycotic activity that Microcyn 60 exhibits in accordance withthis standard is summarized in Table 5.

TABLE 5 Kill Times. Time of action Bacterium Catalog (reduction below99.999%) Ps. aeruginosa ATCC 25619 1 min St. aureus ATCC 6538  1 min E.coli ATCC 11229 1 min S. typhi CDC 99 1 min C. albicans ATCC 1 min B.subtilis 9372 Low spore (10⁴) 10 min  High spore (10⁶) 15 min 

The sporicidal activity trial was carried out in accordance with thePAHO [Pan-American Health Organization]/WHO protocol.

The virucidal activity of Microcyn 60 has recently been confirmed instudies carried out in the United States against HIV and its activityagainst Listeria monocytogenes, MRSA and Mycobacterium tuberculosis hasalso been demonstrated. Thus, it has been demonstrated that Microcyn 60,when it is administered as recommended, can eradicate bacteria, fungi,viruses and spores from one to fifteen minutes of exposure.

Additionally, the following is a clinical study that can be used toassess the efficacy of Microcyn 60 for the treatment ofpharyngitis/tonsilitis. In this study, 40 patients with acutepharyngitis/tonsillitis caused by group A β-hemolytic Streptococcus andwho have not received treatment are recruited. The inclusion criteriaare as follows: age 12 to 40 years and two or more of the followingsymptoms: oropharyngeal burning; pain on swallowing; pharyngeal erythemaor of the tonsils (with or without exudate); cervical lymphadenopathy;and positive immunoassay for group A Streptococcus antigen (StrepATest-Abbott Labs). The exclusion criteria are as follows: fever >38° C.;bronchospasm (excluded by the clinic); severe cough; sinusitis-rhinitis(excluded by the clinic); esophageal reflux (excluded by the clinic);use of antibiotics in the two weeks prior to the study; patients whohave taken part in another clinical study in the last 8 weeks; rheumaticfever; poststreptococcal glomerulonephritis; severe chronic cardiopathy;severe renal, hepatic or pulmonary insufficiencies; and pregnancy orlactation.

At the beginning of the study, patients may use such concomitantmedicines as antipyretics and analgesics, including paracetamol andacetylsalicylics but not anti-inflammatories such as ibuprofen, Mesulid,COX-2 inhibitors, or steroids. Written informed consent must be obtainedbefore the patient submits to any specific procedure of the study.

The patients are evaluated in three visits. In the first visit, thepatient clinically presents acute pharyngitis/tonsillitis, and theclinical history is taken, and a medical examination, rapid immunoassayfor Streptococcus, and taking of a pharyngeal exudate is carried out.After being declared eligible and after having signed the letter ofinformed consent, the patient is prescribed two oropharyngeal cleansingsof 30 sec and 5 mL Microcyn 60 each. These rinsings are done every 3 hfor a total of four times a day for 3 days.

The second is made 72 h after having been treated with Microcyn 60. Inthe second visit, the clinical evolution and side effects of Microcyn 60are evaluated. A new pharyngeal exudate is taken, and it will bedecided, in accordance with the clinical evolution, if the continuingtreatment will be with antibiotics or a palliative. A third visit isdone after 10 days to discharge the patient.

To be eligible and clinically evaluated in this study, each patient mustpresent A β-hemolytic Streptococcus pharyngitis/tonsillitis confirmed byculture. All the patients must comply with 18 rinsings of 30 sec and 5mL of Microcyn 60 each, or a maximum of 24 rinsings in the space of 72h.

The primary parameter of efficacy is a reduction by 3 orders ofmagnitude in the bacterial load of the initial culture compared to theculture taken after the administration of Microcyn 60. Thisbacteriological evaluation is realized 72 h after treatment withMicrocyn 60. Secondary parameters of efficacy are the improvementreported clinically, with particular emphasis on the reduction ofpharyngeal pain and dysphagia. Clinical symptoms are reported in visits1, 2 and 3.

Tolerance is evaluated by reports of adverse events. An adverse event isdefined as any symptomatic declaration of the patient who submits to thetreatment with Microcyn 60, related or not to the antiseptic, thatappears in the course of the treatment.

The results of bacteriological efficacy (the principal criterion ofefficacy) are issued by a bacteriologist independently of the clinicalsymptoms. The tests for the group A Streptococcus antigen and theinitial pharyngeal exudate culture are done in the first visit (Visit1), in accordance with the Schedule of Evaluations and before theadministration of Microcyn 60. The second taking and culture ofpharyngeal exudate is carried out 72 h after the administration ofMicrocyn 60 (Visit 2). An antibiogram is done on all the cultures todetermine the bacterial resistance to penicillin, erythromycin,clarithromycin and lincomycin by means of the standard diffusion disctest. Bacteriological efficacy is defined as the reduction by threeorders of magnitude of the bacterial count between the initial cultureand the culture taken 72 h after administering Microcyn 60.

Bacteriological failure is indicated by a reduction of less than threeorders of magnitude of the bacterial count in the culture at 72 hposttreatment. Indeterminate responses are documented in those cases inwhich the transport of the sample has been delayed for more than 48 h,in those cases in which the swab has not been immersed in the transportmedium, or in those cases in which the sample has been lost. These casesare outside the analysis of the study and are replaced by new casesuntil those of forty eligible patients have been completed.

The follow-up and reporting phase begins when the patient finishes theadministration of Microcyn 60, and from the second visit. In thisevaluation, according to the clinical evolution and the presence ofpossible adverse effects, the patients are categorized as follows:

Therapeutic failures if their initial signs and symptoms have not beeneliminated or if there is worsening of their general condition withsystemic symptoms. In these cases an oral antibiotic is prescribed, suchas procaine penicillin, clarithromycin or azithromycin at the dose andfor the time that the treating doctor indicates, and they are evaluatedin one week.

Clinically cured if the symptoms and signs that were present in Visit 1have been eliminated. In these cases in which the acute process isresolved, the patient is discharged and reported as clinically cured. Inany case, the patient is asked to return for a third check-up visit inone week.

Indeterminate evolution. The evolution of any patient who could not havebeen evaluated clinically for any good reason; for example, acoinfection, or if the evaluation was done very late, later than 72 h.In these cases, the patients is still able to be included in theanalysis of the study provided it is possible to document the result ofthe pharyngeal exudate and culture at 72 h.

The statistical analysis used in this clinical study takes into accountall the patients who have received at least 18 rinsings of Microcyn 60of 30 sec each in a period of 72 h. This same criterion is considered toinclude any patient in the analysis of tolerance. The principalcriterion for analysis of efficacy is the reduction of the bacterialcount of β-hemolytic Streptococcus by three orders of magnitude in theculture carried out at 72 h posttreatment with Microcyn 60. Thestatistical analysis is realized by means of a Wilcoxon paired samplestest. Statistical analysis of the clinical variables is realized usingthe ANOVA test for quantitative variables. The minimal evaluable numberof patients is 30 patients.

An adverse event is any contrary medical occurrence in a patient orsubject of clinical investigation to whom a pharmaceutical product isadministered and that does not necessarily have a causal relationshipwith that medicine. An adverse event can, therefore, be any unfavorableand unintended sign (including an abnormal laboratory finding), symptomor illness temporarily associated with the use of a medical product,whether it is considered to be related to this use or not. Preexistingconditions that deteriorate during a study are reported as adverseevents.

The treatment is suspended at any time during the 72 h of duration incase of adverse events that are moderate to severe in intensity.Subsequent treatment is determined by the treating doctor. In accordancewith this example, the effectiveness of an ORP water solution of thepresent invention for treating sinusitis is thus demonstrated.

EXAMPLE 14

This example demonstrates the viricidal activity of an exemplary ORPwater solution against Adenovirus-serotype 5. For this exampleAdenoviral (Ad) vectors based on human adenovirus type 5 which are E1a-,partially E1-b, and partially E3-deleted were used. A shuttle plasmidcontaining the Green Fluorescent Protein (GFP) reporter gene under thetranscriptional control of pCMV was prepared (pAd-Track). Homologousrecombination of this pShuttle plasmid with AdEasy 1 plasmid was carriedout in electrocompetent bacteria. Clones that had inserts were tested byrestriction endonuclease digestions. Once confirmed, supercoiled plasmidDMA was transformed into DH10B cells for large scale amplification.Subsequently, 293 cells (ATCC 1573) were cultured in serum-free medium(OptiMEM-GIBCO) and transfected with recombinant plasmid digested withPad. Infected cells were monitored for cytopathic effect, collected andlysed with three cycles of freezing and thawing. The resultant viruses(AdGFP) were purified with AdenoPure columns (BD Clontech) according tothe manufacturer's instructions. Viruses were quantitated by OD 260/280.Final yield was 1.52×10¹¹ pfu/mL.

The efficacy of the ORP water solution for inactivating adenovirusencoding the green fluorescence protein gene (AdGFP), was evaluatedusing a test based on the detection of fluorescence emission from HeLacells infected with either, control AdGFP viruses or ORP watersolution-treated AdGFP, using fluorescence-activated flow cytometry.Infection of HeLa cells was always carried out with 7.5×10⁷ pfu/mL (i.e.150 m.o.i.). In all test conditions, cells appeared normal under lightmicroscopy. The background fluorescence measured in control HeLa cellswas 0.06%. After infection with control AdGFP, 88.51% of HeLa cellsexpressed GFP. Following exposure to the ORP water solution, adenovirusinfectivity decreased inversely proportionally to the exposure period.Accordingly, ORP water solution-treated virus for 1, 5, and 10 min couldonly express GFP in 2.8%, 0.13%, and 0.09% of HeLa cell cultures,respectively. Considering the autofluorescence and the initial viralload for all tested conditions (i.e. 7.5×10⁷ pfu), the infectious titerwas 6.6×10⁷ pfu in the control AdGFP-HeLa group. In the groups where thevirus had been treated with the ORP water solution, the infectioustiters were 2.0×10⁶, 5.2×10⁴ and 2.2×10⁴ at one, five and ten minutes ofvirus exposure to the ORP water solution, respectively. Therefore, thelog-10 reduction factor was 1.5, 3.1, and 3.5 at one, five and tenminutes of viral exposure to the ORP water solution. Taken together,these results demonstrate that the virus exposure to the ORP watersolution for 5 minutes achieves a log-10 reduction in the viral load of>3.

EXAMPLE 15

This example demonstrates the viricidal effectiveness of an exemplaryORP water solution against HIV using the United States EnvironmentalProtection Agency protocol for disinfection of inanimate environmentalsurfaces.

The SF33 strain of HIV-1 used for this study. Peripheral bloodmononuclear cells from healthy donors were activated with PHA (3 μg/mL,Sigma) and human IL-2 (20 U/mL, Roche) in HUT media for three days.Cells were washed and infected with SF33 strain. Supernatant wascollected on days 4 and 6, and tested for the p24 HIV-1 antigen by ELISA(Beckman Coulter). Superantant was centrifuged to remove cell and debrisat 3000 RPM for 20 min at room temperature. Supernatant was removed,aliquoted, and the virus was stored at −80° C. until the day of use.

Frozen aliquots were thawed at 37° C. for two minutes immediately priorto its use. Serial logarithmic dilutions (−1 to −5) in HUT medium wereused. Films of virus were prepared by spreading 0.2 ml of virus inoculumuniformly over the bottoms of 55 cm² sterile polystyrene Petri dishes.The virus films were air-dried at room temperature (21° C.) in abiological safety cabinet until they looked visibly dry (20 minutes).(To assure that the virus strain (SF33) was capable of replicating andcausing cytopathic effects, the procedure was repeated with a viralsuspension that had remained in HUT medium without being dried.)

The control film was exposed to 2 ml HUT media for five minutes. Thevirus was then scraped and diluted. Separate dried films were exposed to2 ml each of the ORP water solution for five minutes at roomtemperature. Following the exposure time, the plates were scraped andtheir contents were resuspended. The virus-ORP water solution mixturewas immediately diluted (10:1) in HUT medium. Serial log dilutions ofthis resulting suspension were assayed for infectivity. (To control fora possible direct cytotoxic effect of ORP water solution on MT-2 cells,a 2 ml aliquot of ORP water solution was diluted serially (10:1 to 10:5)in medium and inoculated into MT-2 cell cultures.)

The MT-2 cell line was used as the indicator cell line in theinfectivity assays. This line shows a cytopathic effect consisting ofsincitia formation when infected with HIV-1. Four microwells wereinoculated with 0.2 ml of each dilution of the reconstituted virussuspension from test (reconstituted in ORP water) and control(reconsituted with control medium) groups. Uninfected cell controls wereinoculated with test medium only. Cultures were incubated at 37° C. and5% CO₂.

The cultures were scored periodically every two days for the presence orabsence of cytopathic effect as well as presence of p24-HIV-1 antigen byELISA. Experimental infection with control HIV-1 exerted a cytopathiceffect and Ag p24 protein release into the supernatant in infected MT-2cultures. In contrast, treatment of HIV-1 with the ORP water solutionfor five minutes, achieved a log reduction factor >3 in the viral loadas measured in MT-2 cultures by both assays. These results thusdemonstrate the level of efficacy that is in conformity with the EPArequirements for HIV-1 virucidal activity on inanimate surfaces.

EXAMPLE 16

This example demonstrates the effect of an exemplary ORP water solutionversus hydrogen peroxide (HP) on the viability of human diploidfibroblasts (HDFs). To study this potential toxicity, HDFs were exposedin vitro to ORP water solution and hydrogen peroxide (HP). HP is knownto be toxic to eukaryotic cells, increasing apoptosis and necrosis andreducing cellular viability. In this example, cell viability, apoptosisand necrosis were measured in HDFs exposed to pure ORP water solutionand 880 mM HP (a concentration employed for antiseptic uses of HP) for 5and 30 minutes.

HDF cultures were obtained from three different foreskins, which werepooled and cryopreserved together for the purpose of this study. Onlydiploid cells were used for all experiments. On cell cycle analysis, DNAdiploidy was defined as the presence of a single G0-G1 peak with aCV</=7% and a corresponding G2/M peak collected from at least 20,000total events. FIG. 4A-4C disclose the results where exposure times of 5and 30 minutes are depicted in white and black bars, respectively.Simultaneous analyses of these parameters were performed in the samecell populations by flow cytometry using: A) 7-aminoactinomycin D(7AAD); B) Annexin V-FITC; and C) Propidium iodide. FIG. 4A-4C disclosepercentage values expressed as mean±SD (n=3).

Cell viability was 75% and 55% after a 5 minute exposure to antisepticconcentrations of full strength-ORP water solution and 880 mM HP,respectively (FIG. 4A). The effect of full strength ORP water solutionon cell viability was comparable to a very diluted HP solutionconsidered sublethal but not disinfectant (i.e. 500 μM). If the exposurewas prolonged to 30 min, cell viability further decreased to 70% and 5%,respectively. Apparently, the ORP water solution induced cell deaththrough necrosis because 15% of the cells incorporated propidium iodidein the flow cytometry analysis at both times (FIG. 4C). Apoptosis doesnot seem to be the mechanism by which the ORP water solution inducescell death because only 3% of ORP water solution-treated cells exposedAnnexin-V in the cellular surface (a marker of apoptosis) (FIG. 4B).This percentage was actually similar to the one measured in the controlgroup. On the contrary, HP induced necrosis in 20% and 75% of treatedcells and apoptosis in 15% and 20% after 5 and 30 min of exposure,respectively. Altogether these results show that the (undiluted) ORPwater solution is far less toxic for HDFs than an antisepticconcentration of HP.

EXAMPLE 17

This example demonstrates the effect of an exemplary ORP water solutionrelative to hydrogen peroxide (HP) on oxidative DNA damage and formationof the DNA adduct 8-hydroxy-2′-deoxiguanosine (8-OHdG) in HDFs. It isknown that the production of 8-OHdG adducts in a cell is a marker ofoxidative damage at specific residues of DNA. In addition, high cellularlevels of this adduct correlate with mutagenesis, carcinogenesis andcellular aging.

FIG. 5 shows the levels of 8-OHdG adducts present in DNA samples fromHDFs after control treatments, ORP water solution treatments andHP-treatments for 30 minutes. DNA was extracted right after the exposure(T0, white bars) or three hours after the challenge period (T3, blackbars). DNA was digested and the 8-OHdG adducts were measured by ELISAkit as per the manufacturer's instructions. Values are shown (ng/mL) asmean±SD (n=3). The exposure to ORP water solution for 30 minutes did notincrease the formation of adducts in the treated cells in comparison tocontrol cells after incubation for 30 minutes. In contrast, thetreatment with 500 μM HP for 30 minutes increased the number of 8-OHdGadducts by about 25 fold relative to the control-treated or ORP watersolution-treated cells.

The ORP water solution-treated cells were able to decrease the levels of8-OHdG adducts if left in supplemented DMEM for 3 hours after exposureto the ORP water solution. Despite being allowed the same 3 hourrecovery period, HP-treated cells still presented about 5 times moreadducts than control-treated or ORP water solution treated cells.Altogether, these results demonstrate that acute exposure to the ORPwater solution does not induce significant DNA oxidative damage. Theseresults also indicate that the ORP water solution will not likely inducemutagenesis or carcinogenesis in vitro or in vivo.

EXAMPLE 18

This example demonstrates the effects on HDFs of chronic exposure to lowconcentrations of an exemplary ORP water solution versus HP. It is knownthat chronic oxidative stress induces premature aging of cells. In orderto mimick a prolonged oxidative stress, primary HDF cultures werechronically exposed to low concentrations of the ORP water solution(10%) or HP (5 μM) during 20 population doublings. The expression andactivity of the SA-β-galactosidase enzyme has previously been associatedwith the senescence process in vivo and in vitro. In this example theexpression of the SA-β-galactosidase enzyme was analyzed after one monthof continuous exposure of HDF to the ORP water solution or HP. Theresults are depicted in FIG. 6. The expression of the enzymeSA-β-galactosidase was analyzed by counting the number of blue cells in20 microscopic fields. FIG. 6 shows that only HP treatment acceleratedthe aging of cells as indicated by the number of cells over-expressingSA-β-galactosidase (n=3). Chronic treatment with a low dose of HPincreased the SA-β-Gal expression in 86% of cells while the treatmentwith the ORP water solution did not induce the overexpression of thisprotein. It can be concluded from this example that ORP water solutionis not an inducer of premature cellular aging.

EXAMPLE 19

This example demonstrates the effect of an exemplary ORP water solutionon the reduction of peritoneal bacterial load and on the reduction inthe length of hospital stay in patients with peritonitis. All patientsadmitted to the Hospital Ruben Leñero in Mexico City from June 2004 toJanuary 2005, and with a diagnosis of acute generalized,secondaryperitonitis, were included in the ORP water solution-treated group.Secondary peritonitis was defined as the result of the loss of integrityof the gastrointestinal or genito-urinary tract leading to contaminationof the peritoneal space. Retrospective analysis of paired-casespresenting similar peritoneal infections between 2003 and 2004 at thesame Institution was undertaken for the control group. Twentyconsecutive patients were prospectively included in the ORP watersolution-treated group (i.e. study group).

Upon admission, all patients underwent open surgery and intra-operativeperitoneal lavage (“IOPL”) of all quadrants of the abdomen.Intraoperative peritoneal-culture samples were taken in both groups.IOPL was performed with 10 L of saline solution in both groups andfollowed by 5 L of the ORP water solution in the study group only. Theexcess ORP water solution was removed and no further rinsing wasconducted. The abdominal cavity was covered with a plastic mesh in bothgroups. However, in the study group, a dressing soaked in ORP watersolution was left on top of the mesh. The dressing was changed t.i.d.Emperic antimicrobial therapy was started in all patients with twoantibiotics including clindamycin and cefotaxime or amikacin.Post-operative management in the study group included daily irrigationof the mesh with 100 mL of the ORP water solution t.i.d., withoutfurther rinsing or lavage. Severe cases of peritonitis requiredre-laparotomy and IOPL every 72 hours. Cultures of the peritoneal fluidfor aerobic bacteria and fungi were taken every 72 hours in both groupsfor up to one week. The duration of length of stay in the hospital wasrecorded.

Twenty control cases were selected from the medical records of theInstitution and paired to the study group by age, sex and etiology ofperitonitis. The control and study populations were comparable in age,sex and prognostic factors at entry. The anatomic origin and etiology ofperitonitis was also similar for both groups (Table 6).

TABLE 6 Diagnoses. DIAGNOSIS CONTROL STUDY TOTAL % Appendicitis 3 6 923.0 Post-trauma 1 3 4 10.0 Pancreatitis 6 3 9 23.0 Cholecystitis 1 2 37.5 Colon cancer 0 1 1 2.5 Small bowel 4 1 5 12.5 fistula Diverticulitis1 1 2 5.0 Gastric 4 0 4 10.0 perforation Other Organ 0 2 2 5.0perforation Other 0 1 1 2.5 TOTAL 20 20 40 100.0

Post-operative peritonitis was present in 19 and 17 patients of thecontrol and study groups, respectively. All cases underwent surgicaltreatment followed by IOPL. The types of surgeries performed incontrol/study groups, were: appendicectomy (3/6), gastric resection(4/0), cholecystectomy (1/2), pancreatic necrosectomy (6/3), small bowelsuture/resection with anastomosis (4/3), Hartman's operation (1/1),colonic resection (0/1) and miscellaneous (1/4). The use of antibioticswas very similar in both groups. For control and study groups, threeantibiotics were administered in 16 and 15 patients and more than 3antibiotics in 4 and 5 cases, respectively. Patients were kept at theICU and were mechanically ventilated post-operatively. Peri-operativeintra-abdominal samples were taken in all 40 patients (Table 7).

TABLE 7 Microorganisms isolated from intraperitoneal samples and lengthof hospital stay in patients with peritonitis. CONTROL GROUP STUDY GROUPIsolated Isolated strains (n) Hospital strains (n) Hospital OrganismPeri-op Post-op Days Peri-op Post-op Days C. albicans 10 7 19.4 7 0 6.3E. coli 3 2 17.6 6 1 10.2 S. aureus 10 9 22.3 8 1 14.1 coagulase 0 0 0 20 17.8 neg. Staph. A. baumanii 0 0 0 1 0 22.4 E. faecalis 3 3 23.7 1 028.6 A. 0 0 0 1 0 28.6 xilosoxidans P. 2 2 24.0 3 0 33.9 aeruginosa E.coacae 1 1 13.0 1 0 37.0 TOTAL 29 24 31.9 30 2 22.4

Samples were obtained in the peri-operative period and in the followingweek after intra-operative lavage with saline solution only (controlgroup) or saline solution and ORP water solution (study group). Theaverage hospital stay was then analyzed for each microorganism isolatedat entry and for the whole group.

Peri-operative samples were taken in all 40 patients (Table 7). Theaverage numbers of microorganisms grown from these samples were 29 inthe control and 30 in the study group. The microorganisms isolated areshown in Table 8. Escherichia coli, Enterococcus, Staphylococcus aureus,Pseudomonas aeruginosa and fungi were isolated from these groups in 3/6,4/2, 10/8, 2/3 and 10/7 occasions, respectively. Positive cultures forA. xilosoxidans (1), coagulase negative Staphylococci (2) and A.baumanii (1) were only found in the study group.

A second intra-abdominal culture was taken during the first week aftersurgery (Table 7). At this time, the average number of organismsisolated in the control group (24) was almost the same as in theperi-operative sample (29). Importantly, there was a strong reduction inthe number of positive samples in the study group. From 30 positivecultures in the peri-operative samples, only one remained positive forS. aureus and another one for E. coli. In the analysis of hospital days,the control group had a longer stay (31.9 days) in comparison to thestudy group (22.4 days). Thus, the ORP water solution effectivelyreduced the peritoneal bacterial load and length of hospital stay inpatients with peritonitis.

The mortality rates were also analyzed. There were six deaths in thecontrol group and 3 in the study one. All deaths occurred in the first30 days after the first surgery and the calculated relative risk washigher for the control group (i.e. 3.3 versus 0). However, the samplesize was too small to achieve statistical significance. No local sideeffects were recorded with the use of ORP water in the IOPL. Survivingpatients in the study group were followed for 6 to 12 months. None ofthe 20 patients in the ORP water-treated group presented intestinalocclusion or data suggesting sclerosing peritonitis in the follow-upperiod.

EXAMPLE 20

This example demonstrates the effectiveness of an exemplary ORP watersolution (Mycrocyn) in inhibiting mast cell degranulation. Mast cellshave been recognized as principal players in type I hypersensitivitydisorders. Multiple clinical symptoms observed in atopic dermatitis,allergic rhinitis, and atopic asthma are produced by IgE-antigenstimulation of mast cells located in distinct affected tissues. Thecurrently accepted view of the pathogenesis of atopic asthma is thatallergens initiate the process by triggering IgE-bearing pulmonary mastcells (MCs) to release mediators such as histamine, leukotrienes,prostaglandins, kininis, platelet activating factor (PAF), etc. in theso-called early phase of the reaction. In turn, these mediators inducebronchoconstriction and enhance vascular permeability and mucusproduction. According to this model, following mast cell activation,those cells secrete various pro-inflammatory cytokines in a late phase,including tumor necrosis factor alpha (TNF-α), IL-4, IL-5 and IL-6,which participate in the local recruitment and activation of otherinflammatory cells such as eosinophils, basophils, T lymphocytes,platelets and mononuclear phagocytes. These recruited cells, in turn,contribute to the development of an inflammatory response that may thenbecome autonomous and aggravate the asthmatic symptoms. This late phaseresponse constitutes a long term inflammation process which can induceplastic changes in surrounding tissues (see Kumar et al., pp. 193-268).

Antigenic stimulation of mast cells occurs via the activation of thehigh affinity receptor for IgE (the FcεRI receptor), which is amultimeric protein that binds IgE and subsequently can be aggregated bythe interaction of the receptor-bound IgE with a specific antigen. Itsstructure comprises four polypeptides, an IgE binding α chain, a β chainthat serves to amplify its signaling capacity, and two disulfide-linkedγ chains, which are the principal signal transducers via the encodedimmunoreceptor tyrosine-based (ITAM) activation motif. Signalingpathways activated by the cross-linking of this receptor have beencharacterized using bone marrow-derived mast cells (BMMC), the ratleukemia cell line RBL 2H3, mouse and rat peritoneal mast cells, andother mast cell lines, such as MC-9, In all of them, the presence ofantigen bound to IgE causes mast cell degranulation, calciummobilization, cytoskeletal re-arrangements and activation of differenttranscription factors (NFAT, NFκB, AP-1, PU.1, SP1, Ets, etc.) whichactivate cytokine gene transcription that culminate with cytokineproduction.

Mature murine bone-derived mast cells (BMMC) were loaded with amonoclonal anti-Dinitrophenol IgE (300 ng/million cell) during 4 hoursat 37° C. Culture media was removed and cells were resuspended inphysiological buffer (Tyrode's Buffer/BSA). Cells were then treated 15minutes at 37° C. with distinct concentrations of the ORP water solution(in its Microcyn embodiment). Buffer was removed and cells resuspendedin fresh Tyrode's/BSA and stimulated with different concentrations ofantigen (Human Albumin coupled to Dinitrophenol) during a 30 minuteincubation at 37° C. Degranulation was measured by β-hexosaminidaseactivity determination in supernatants and pellets of the stimulatedcells, using a colorimetric reaction based on the capacity of thisenzyme to hydrolize distinct carbohydrates. (β-hexosaminidase has beenshown to be located in the same granules that contain histamine in mastcells.) The results (FIG. 7) demonstrate that degranulation issignificantly reduced with increasing concentrations of the ORP watersolution.

Surprisingly, the inhibitory effect of the ORP water solution (Microcyn)on mast cell degranulation at least is similar to that observed with theclinically effective “mast cell stabilizer” and establishedanti-allergic compound sodium cromoglycate (Intel™) Degranulation wasagain measured by β-hexosaminidase enzymatic activity in the pellet andsupernatant of stimulated cells, using a colorimetric reaction based onthe capacity of this enzyme to hydrolize distinct carbohydrates. Cellsloaded with anti-DNP monoclonal IgE were stimulated with or without a 15minute pre-incubation with sodium cromoglycate (Intel™). Cromoglycatewas no more effective than the ORP water solution in reducingdegranulations (Compare FIG. 7 with FIG. 8; both achieving at leastabout 50% reduction in degranulation.)

EXAMPLE 21

This example demonstrates the inhibitory activity of an exemplary ORPwater solution on mast cell activation by a calcium ionophore.

Mast cells can be stimulated via the activation of calcium fluxesinduced by a calcium ionophore. Signaling pathways activated by calciumionophores have been characterized using bone marrow-derived mast cells(BMMC), the rat leukemia cell line RBL 2H3, mouse and rat peritonealmast cells, and other mast cell lines, such as MC-9. In all of thesesystems the calcium mobilization causes mast cell degranulation (e.g.histamine release), cytoskeletal re-arrangements, and activation ofdifferent transcription factors (e.g., NFAT, NFκB, AP-1, PU.1, SP1,Ets.) which activate cytokine gene transcription that culminate withcytokine production and secretion.

Mature murine BMMC were loaded with a monoclonal anti-Dinitrophenol IgE(300 ng/million cell) during 4 hours at 37° C. Culture media was removedand cells were resuspended in physiological buffer (Tyrode'sBuffer/BSA). Cells were then treated for 15 minutes at 37° C. withdistinct concentrations of the ORP water solution (Microcyn). Buffer wasremoved and cells were resuspended in fresh Tyrode's/BSA and stimulatedwith calcium ionophore (100 mM A23187) during a 30 minute incubation at37° C. Degranulation was measured by β-hexosaminidase activitydetermination in supernatants and pellets of the stimulated cells, usinga colorimetric reaction based on the capacity of this enzyme tohydrolyze distinct carbohydrates. (β-hexosaminidase has been shown to belocated in the same granules that contain histamine in mast cells.) Theresults (FIG. 8) demonstrate that degranulation is significantly reducedwith increasing concentrations of the ORP water solution.

These results suggest that ORP water solution is a non-specificinhibitor of histamine release. Thus, ORP water solution -even atdifferent concentrations- will inhibit the degranulation of mast cellsindependently of the stimulus (e.g. antigen or ionophore). While notdesiring to be bound by any theory, ORP water solution probably modifiesthe secretory pathway system at the level of the plasma membrane and/orcytoskeleton. Because the mechanism of action of ORP water solution isbelieved to be non-specific, it is believed that ORP water solution canhave broad potential clinical applications.

EXAMPLE 22

This example demonstrates the effect of an exemplary ORP water solutionon the activation of mast cell cytokine gene transcription.

FIGS. 10A and 10B are RNAase protection assays from mast cells treatedwith ORP water solution at different concentrations for 15 minutes andfurther stimulated by antigen as described in Example 20. Afterstimulation, mRNA was extracted using affinity chromatography columns(RNAeasy kit, Qiagene) and the RNAse Protection Assay was performedusing standard kit conditions (Clontech, Becton & Dickinson) in order todetect mRNA production of distinct cytokines after antigen challenge.The cytokines included TNF-α, LIF, IL13, M-CSF, IL6, MIF and L32.

FIGS. 10A and 10B show that the ORP solution water (Microcyn) did notmodify cytokine mRNA levels after antigen challenge in mast cellsirrespective of the concentrations of ORP water solution or antigen usedfor the experiment.

In this study, the level of transcripts (i.e., the RNA content ofstimulated mast cells) of proinflammatory genes was not changed in ORPwater solution-treated mast cells after being stimulated with variousconcentrations of antigen. Thus, the ORP water solution inhibited thesecretory pathway of these cytokines without affecting theirtranscription.

EXAMPLE 23

This example demonstrates the inhibitory activity of an exemplary ORPwater solution on mast cell secretion of TNF-α.

Mast cells were treated with different concentrations of ORP watersolution for 15 minutes and further stimulated by antigen as describedin Example 20, Thereafter, the tissue culture medium was replaced andsamples of the fresh medium were collected at various periods of time(2-8 hours) for measuring TNF-α levels. Samples were frozen and furtheranalyzed with a commercial ELISA kit (Biosource) according to themanufacturer's instructions.

FIG. 11 shows that the level of secreted TNF-α to the medium from ORPwater solution-treated cells after antigen stimulation is significantlydecreased in comparison to the untreated cells.

Since the release of TNF-α and that of various other pro-inflammatorymolecules depends on a separate secretory pathway than that ofhistamine, it is possible that the ORP solution can stop the secretionof those other cytokines leading the late inflammatory phase.

Thus, the ORP water solution inhibited TNF-α secretion ofantigen-stimulated mast cells. These results are in agreement withclinical observations that the use of ORP water solutions can decreasethe inflammatory reaction in various wounds after surgical procedures.

EXAMPLE 24

This example demonstrates the inhibitory activity of an exemplary ORPwater solution on mast cell secretion of MIP 1-α.

Mast cells were treated with different concentrations of an exemplaryORP water solution (Microcyn) for 15 minutes and further stimulated byantigen as described in Example 20. Thereafter, the tissue culturemedium was replaced and samples of the fresh medium were collected atvarious periods of time (2-8 hours) for measuring MIP 1-α levels.Samples were frozen and further analyzed with a commercial ELISA kit(Biosource) according to the manufacturer's instructions.

FIG. 12 shows that the level of secreted MIP 1-α to the medium from ORPwater solution-treated cells after antigen stimulation was significantlydecreased in comparison to the untreated cells.

Thus, the ORP water solution inhibited MIP 1-α secretion ofantigen-stimulated mast cells. These results are in agreement withclinical observations that the use of ORP water solutions can decreasethe inflammatory reaction in various wounds after surgical procedures.

Since the release of MIP 1-α and that of various other pro-inflammatorymolecules depends on a separate secretory pathway than that ofhistamine, it is possible that the ORP solution can stop the secretionof those other cytokines leading the late inflammatory phase.

The results of analogous studies measuring IL-6 and IL-13 secretion aredepicted in FIGS. 13 and 14.

Examples 20-23 and this example further demonstrate that the ORP watersolution is able to inhibit early and late phase allergic responsesinitiated by IgE receptor crosslinking.

EXAMPLE 25

This example demonstrates the results of a toxicity study using anexemplary ORP water solution.

An acute systemic toxicity study was performed in mice to determine thepotential systemic toxicity of Microcyn 60, an exemplary ORP watersolution. A single dose (50 mL/kg) of Microcyn 60 was injectedintraperitoneally in five mice. Five control mice were injected with asingle dose (50 mL/kg) of saline (0.9% sodium chloride). All animalswere observed for mortality and adverse reactions immediately followingthe injection, at 4 hours after injection, and then once daily for 7days. All animals were also weighed prior to the injection and again onDay 7. There was no mortality during the study. All animals appearedclinically normal throughout the study. All animals gained weight. Theestimated Microcyn 60 acute intraperitoneal LD50 from this study isgreater than 50 mL/kg. This example demonstrates that Microcyn 60 lackssignificant toxicity and should be safe for therapeutic use accordancewith the invention.

EXAMPLE 26

This example illustrates a study conducted to determine the potentialcytogenetic toxicity of an exemplary ORP water solution.

A micronucleus test was performed using an exemplary ORP water solution(10% Microcyn™) to evaluate the mutagenic potential of intraperitonealinjection of an ORP water solution into mice. The mammalian in vivomicronucleus test is used for the identification of substances whichcause damage to chromosomes or the mitotic apparatus of murinepolychromatic erythrocytes. This damage results in the formation of“micronuclei,” intracellular structures containing lagging chromosomefragments or isolated whole chromosomes. The ORP water solution studyincluded 3 groups of 10 mice each (5 males/5 females): a test group,dosed with the ORP water solution; a negative control group, dosed witha 0.9% NaCl solution; and a positive control group, dosed with amutagenic cyclophosphamide solution. The test and the negative controlgroups received an intraperitoneal injection (12.5 ml/kg) of the ORPwater solution or 0.9% NaCl solution, respectively, for two consecutivedays (days 1 and 2). The positive control mice received a singleintraperitoneal injection of cyclophosphamide (8 mg/mL, 12.5 ml/kg) onday 2. All mice were observed immediately after injection for anyadverse reactions. All animals appeared clinically normal throughout thestudy and no sign of toxicity was noted in any group. On day 3, all micewere weighed and terminated.

The femurs were excised from the terminated mice, the bone marrow wasextracted, and duplicate smear preparations were performed for eachmouse. The bone marrow slides for each animal were read at 40×magnification. The ratio of polychromatic erythrocytes (PCE) tonormochromatic erythrocytes (NCE), an index of bone marrow toxicity, wasdetermined for each mouse by counting a total of at least 200erythrocytes. Then a minimum of 2000 scoreable PCE per mouse wereevaluated for the incidence of micronucleated polychromaticerythrocytes. Statistical analysis of the data were done using the Mannand Whitney test (at 5% risk threshold) from a statistical softwarepackage (Statview 5.0®, SAS Institute Inc., USA).

The positive control mice had statistically significant lower PCE/NCEratios when compared to their respective negative controls (males: 0.77vs. 0.90 and females: 0.73 vs. 1.02), showing the toxicity of thecyclophosphamide on treated bone marrow. However, there was nostatistically significant difference between the PCE/NCE ratios for theORP water solution-treated mice and negative controls. Similarly,positive control mice had a statistically significant higher number ofpolychromatic erythrocytes bearing micronuclei as compared to both theORP water solution-treated mice (males: 11.0 vs. 1.4/females: 12.6 vs.0.8) and the negative controls (males: 11.0 vs. 0.6/females: 12.6 vs.1.0). There was no statistically significant difference between thenumber of polychromatic erythrocytes bearing micronculei in ORP watersolution-treated and negative control mice.

This example demonstrates that Microcyn™ 10% did not induce toxicity ormutagenic effects after intraperitoneal injections into mice.

EXAMPLE 27

This study demonstrates the lack of toxicity of an exemplary ORP watersolution, Dermacyn.

This study was done in accordance with ISO 10993-5:1999 standard todetermine the potential of an exemplary ORP water solution, Dermacyn, tocause cytotoxicity. A filter disc with 0.1 mL of Dermacyn was placedonto an agarose surface, directly overlaying a monolayer of mousefibroblast cells (L-929). The prepared samples were observed forcytotoxic damage after 24 hours of incubation at 37° C. in the presenceof 5% CO₂. Observations were compared to positive and negative controlsamples. The Dermacyn containing samples did not reveal any evidence ofcell lysis or toxicity, while positive and negative control performed asanticipated.

Based on this study Dermacyn was concluded not to generate cytotoxiceffects on murine fibroblasts.

EXAMPLE 28

This study was conducted with 16 rats to evaluate the local tolerabilityof an exemplary ORP water solution, Dermacyn, and its effects on thehistopathology of wound beds in a model of full-thickness dermal woundhealing. Wounds were made on both sides of the subject rat. During thehealing process skin sections were taken on either the left or the rightsides (e.g., Dermacyn-treated and saline-treated, respectively).

Masson's trichrome-stained sections and Collagen Type II stainedsections of the Dermacyn and saline-treated surgical wound sites wereevaluated by a board-certified veterinary pathologist. The sections wereassessed for the amount of Collagen Type 2 expression as amanifestiation of connective tissue proliferation, fibroblast morphologyand collagen formation, presence of neoepidermis in cross section,inflammation and extent of dermal ulceration.

The findings indicate that Dermacyn was well tolerated in rats. Therewere no treatment-related histopathologic lesions in the skin sectionsfrom either sides' wounds (Dermacyn-treated and saline-treated,respectively). There were no relevant histopathologic differencesbetween the saline-treated and the Dermacyn-treated wound sites,indicating that the Dermacyn-treatement was well tolerated. There wereno significant differences between Collagen Type 2 expression betweenthe saline-treated and the Dermacyn™-treated wound sites indicating thatthe Dermacyn does not have an adverse effect on fibroblasts or oncollagen elaboration during wound healing.

EXAMPLE 29

This example demonstrates the use of an exemplary oxidative reductivepotential water, Microcyn, in accordance with the invention as aneffective antimicrobial solution.

An In-Vitro Time-Kill evaluation was performed using Microcyn oxidativereductive potential water. Microcyn was evaluated versus challengesuspensions of fifty different microorganism strains—twenty-fiveAmerican Type Culture Collection (ATCC) strains and twenty-five ClinicalIsolates of those same species—as described in the Tentative FinalMonograph, Federal Register, 17 Jun. 1994, vol. 59:116, pg. 31444. Thepercent reductions and the Log₁₀ reductions from the initial populationof each challenge strain were determined following exposures to Microcynfor thirty (30) seconds, one (1) minute, three (3) minutes, five (5)minutes, seven (7) minutes, nine (9) minutes, eleven (11) minutes,thirteen (13) minutes, fifteen (15) minutes, and twenty (20) minutes.All agar-plating was performed in duplicate and Microcyn was evaluatedat a 99% (v/v) concentration. All testing was performed in accordancewith Good Laboratory Practices, as specified in 21 C.F.R. Part 58.

The following table summarizes the results of the abovementionedIn-Vitro Time-Kill evaluation at the thirty second exposure mark for allpopulations tested which were reduced by more than 5.0 Log₁₀:

TABLE 8 30-Second In-Vitro Kill. Initial Post-Exposure PopulationPopulation Log₁₀ Percent No. Microorganism Species (CFU/mL) (CFU/mL)Reduction Reduction 1 Acinetobacter baumannii  2.340 × 10⁹  <1.00 × 10³6.3692 99.9999 (ATCC #19003) 2 Acinetobacter baumannii 1.8150 × 10⁹ <1.00 × 10³ 6.2589 99.9999 Clinical Isolate BSLI #061901Ab3 3Bacteroides fragilis  4.40 × 10¹⁰ <1.00 × 10³ 7.6435 99.9999 (ATCC#43858) 4 Bacteroides fragilis  2.70 × 10¹⁰ <1.00 × 10³ 7.4314 99.9999Clinical Isolate BSLI #061901Bf6 5 Candida albicans  2.70 × 10¹⁰ <1.00 ×10³ 6.3345 99.9999 (ATCC #10231) 6 Candida albicans  5.650 × 10⁹  <1.00× 10³ 6.7520 99.9999 Clinical Isolate BSLI #042905Ca 7 Enterobacteraerogenes 1.2250 × 10⁹  <1.00 × 10³ 6.0881 99.9999 (ATCC #29007) 8Enterobacter aerogenes 1.0150 × 10⁹  <1.00 × 10³ 6.0065 99.9999 ClinicalIsolate BSLI #042905Ea 9 Enterococcus faecalis  2.610 × 10⁹  <1.00 × 10³6.4166 99.9999 (ATCC #29212) 10 Enterococcus faecalis 1.2850 × 10⁹ <1.00 × 10³ 6.1089 99.9999 Clinical Isolate BSLI #061901Efs2 11Enterococcus faecium  3.250 × 10⁹  <1.00 × 10³ 6.5119 99.9999 VRE, MDR(ATCC #51559) 12 Enterococcus faecium  1.130 × 10⁹  <1.00 × 10³ 6.053199.9999 Clinical Isolate BSLI #061901Efm1 13 Escherichia coli  5.00 ×10⁸  <1.00 × 10³ 5.6990 99.9998 (ATCC #11229) 14 Escherichia coli  3.950× 10⁸  <1.00 × 10³ 5.5966 99.9997 Clinical Isolate BSLI #042905Ec1 15Escherichia coli  6.650 × 10⁸  <1.00 × 10³ 5.8228 99.9998 (ATCC #25922)16 Escherichia coli  7.40 × 10⁸  <1.00 × 10³ 5.8692 99.9998 ClinicalIsolate BSLI #042905Ec2 17 Haemophilus influenzae 1.5050 × 10⁹  <1.00 ×10⁴ 5.1775 99.9993 (ATCC #8149) 18 Haemophilus influenzae  1.90 × 10⁹ <1.00 × 10⁴ 5.2788 99.9995 Clinical Isolate BSLI #072605Hi 19 Klebsiellaoyxtoca  1.120 × 10⁹  <1.00 × 10³ 6.0492 99.9999 MDR (ATCC #15764) 20Klebsiella oyxtoca  1.810 × 10⁹  <1.00 × 10³ 6.2577 99.9999 ClinicalIsolate BSLI #061901Ko1 21 Klebsiella pneumoniae  1.390 × 10⁹  <1.00 ×10³ 6.1430 99.9999 subsp. ozaenae (ATCC #29019) 22 Klebsiella pneumoniae 9.950 × 10⁸  <1.00 × 10³ 5.9978 99.9999 Clinical Isolate BSLI#061901Kpn2 23 Micrococcus luteus  6.950 × 10⁸  <1.00 × 10³ 5.842099.9999 (ATCC #7468) 24 Micrococcus luteus 1.5150 × 10⁹  <1.00 × 10³6.1804 99.9999 Clinical Isolate BSLI #061901M12 25 Proteus mirabilis1.5950 × 10⁹  <1.00 × 10³ 6.2028 99.9999 (ATCC #7002) 26 Proteusmirabilis 2.0950 × 10⁹  <1.00 × 10³ 6.3212 99.9999 Clinical Isolate BSLI#061901Pm2 27 Pseudomonas aeruginosa  6.450 × 10⁸  <1.00 × 10³ 5.809699.9999 (ATCC #15442) 28 Pseudomonas aeruginosa 1.3850 × 10⁹  <1.00 ×10³ 6.1414 99.9999 Clinical Isolate BSLI #072605Pa 29 Pseudomonasaeruginosa  5.550 × 10⁸  <1.00 × 10³ 5.7443 99.9999 (ATCC #27853) 30Pseudomonas aeruginosa 1.1650 × 10⁹  <1.00 × 10³ 6.0663 99.9999 ClinicalIsolate BSLI #061901Pa2 31 Serratia marcescens  9.950 × 10⁸  <1.00 × 10³5.9978 99.9999 (ATCC #14756) 32 Serratia marcescens 3.6650 × 10⁹  <1.00× 10³ 6.5641 99.9999 Clinical Isolate BSLI #042905Sm 33 Staphylococcusaureus 1.5050 × 10⁹  <1.00 × 10³ 6.1775 99.9999 (ATCC #6538) 34Staphylococcus aureus  1.250 × 10⁹  <1.00 × 10³ 6.0969 99.9999 ClinicalIsolate BSLI #061901Sa1 35 Staphylococcus aureus  1.740 × 10⁹  <1.00 ×10³ 6.2405 99.9999 (ATCC #29213) 36 Staphylococcus aureus  1.1050 × 10⁹ <1.00 × 10³ 6.0434 99.9999 Clinical Isolate BSLI #061901Sa2 37Staphylococcus epidermidis 1.0550 × 10⁹  <1.00 × 10³ 6.0233 99.9999(ATCC #12228) 38 Staphylococcus epidermidis  4.350 × 10⁸  <1.00 × 10³5.6385 99.9998 Clinical Isolate BSLI #072605Se 39 Staphylococcushaemolyticus  8.150 × 10⁸  <1.00 × 10³ 5.9112 99.9999 (ATCC #29970) 40Staphylococcus haemolyticus  8.350 × 10⁸  <1.00 × 10³ 5.9217 99.9999Clinical Isolate BSLI #042905Sha 41 Staphylococcus hominis  2.790 × 10⁸ <1.00 × 10³ 5.4456 99.9996 (ATCC #27844) 42 Staphylococcus hominis  5.20× 10⁸  <1.00 × 10³ 5.7160 99.9998 Clinical Isolate BSLI #042905Sho 43Staphylococcus saprophyticus  9.10 × 10⁸  <1.00 × 10³ 5.9590 99.9999(ATCC #35552) 44 Staphylococcus saprophyticus 1.4150 × 10⁹  <1.00 × 10³6.1508 99.9999 Clinical Isolate BSLI #042905Ss 45 Streptococcuspneumoniae 2.1450 × 10⁹  <1.00 × 10⁴ 5.3314 99.9995 (ATCC #33400) 46Streptococcus pyogenes  5.20 × 10⁹  <1.00 × 10³ 6.7160 99.9999 (ATCC#19615) 47 Streptococcus pyogenes Clinical Isolate 2.5920 × 10⁹  <1.00 ×10³ 6.4141 99.9999 BSLI #061901Spy7

While their microbial reductions were measured at less than 5.0 Log₁₀,Microcyn also demonstrated antimicrobial activity against the remainingthree species not included in Table 8. More specifically, a thirtysecond exposure to Microcyn reduced the population of Streptococcuspneumoniae (Clinical Isolate; BSLI #072605Spn1) by more than 4.5 Log₁₀,which was the limit of detection versus this species. Further, whenchallenged with Candida tropicalis (ATCC #750), Microcyn demonstrated amicrobial reduction in excess of 3.0 Log₁₀ following a thirty secondexposure. Additionally, when challenged with Candida tropicalis (BSLI#042905Ct), Microcyn demonstrated a microbial reduction in excess of 3.0Log₁₀ following a twenty minute exposure.

The exemplary results of this In-Vitro Time-Kill evaluation demonstratethat Microcyn oxidative reductive potential water exhibits rapid (i.e.,less than 30 seconds in most cases) antimicrobial activity versus abroad spectrum of challenging microorganisms. Microbial populations offorty-seven out of the fifty Gram-positive, Gram-negative, and yeastspecies evaluated were reduced by more than 5.0 Log₁₀ within thirtyseconds of exposure to the product.

EXAMPLE 30

This example demonstrates a comparison of the antimicrobial activity ofan exemplary oxidative reductive potential water, Microcyn, used inaccordance with the invention versus HIBICLENS® chlorhexidine gluconatesolution 4.0% (w/v) and 0.9% sodium chloride irrigation (USP).

An In-Vitro Time-Kill evaluation was performed as described in Example29 using HIBICLENS® chlorhexidine gluconate solution 4.0% (w/v) and asterile 0.9% sodium chloride irrigation solution (USP) as referenceproducts. Each reference product was evaluated versus suspensions of theten American Type Culture Collection (ATCC) strains specifically denotedin the Tentative Final Monograph. The data collected was then analyzedagainst the Microcyn microbial reduction activity recorded in Example29.

Microcyn oxidative reductive potential water reduced microbialpopulations of five of the challenge strains to a level comparable tothat observed for the HIBICLENS® chlorhexidine gluconate solution. BothMicrocyn and HIBICLENS® provided a microbial reduction of more than 5.0Log₁₀ following a thirty second exposure to the following species:Escherichia coli (ATCC #11229 and ATCC #25922), Pseudomonas aeruginosa(ATCC #15442 and ATCC #27853), and Serratia marcescens (ATCC #14756).Further, as shown above in Table 9, Microcyn demonstrated excellentantimicrobial activity against Micrococcus luteus (ATCC #7468) byproviding a 5.8420 Log₁₀ reduction after a thirty second exposure.However, a direct Micrococcus luteus (ATCC #7468) activity comparison toHIBICLENS® was not possible because after a thirty second exposure,HIBICLENS® reduced the population by the detection limit of the test (inthis specific case, by more than 4.8 Log₁₀). It is noted that thesterile 0.9% sodium chloride irrigation solution reduced microbialpopulations of each of the six challenge strains discussed above by lessthan 0.3 Log₁₀ following a full twenty minute exposure.

Microcyn oxidative reductive potential water provided greaterantimicrobial activity than both HIBICLENS® and the sodium chlorideirrigation for four of the challenge strains tested: Enterococcusfaecalis (ATCC #29212), Staphylococcus aureus (ATCC #6538 and ATCC#29213), and Staphylococcus epidermidis (ATCC #12228). The followingtable summarizes the microbial reduction results of the In-VitroTime-Kill evaluation for these four species:

TABLE 9 Comparative Results Micro- Log₁₀ Reduction organism ExposureNaCl Species Time Microcyn HIBICLENS ® Irrigation Enterococcus 30seconds 6.4166 1.6004 0.3180 faecalis  1 minute  6.4166 2.4648 0.2478(ATCC #29212)  3 minutes 6.4166 5.2405 0.2376  5 minutes 6.4166 5.41660.2305  7 minutes 6.4166 5.4166 0.2736  9 minutes 6.4166 5.4166 0.289511 minutes 6.4166 5.4166 0.2221 13 minutes 6.4166 5.4166 0.2783 15minutes 6.4166 5.4166 0.2098 20 minutes 6.4166 5.4166 0.2847Staphylococcus 30 seconds 6.1775 1.1130 0.0000 aureus  1 minute  6.17751.7650 0.0191 (ATCC #6538)  3 minutes 6.1775 4.3024 0.0000  5 minutes6.1775 5.1775 0.0000  7 minutes 6.1775 5.1775 0.0000  9 minutes 6.17755.1775 0.0000 11 minutes 6.1775 5.1775 0.0267 13 minutes 6.1775 5.17750.0000 15 minutes 6.1775 5.1775 0.0191 20 minutes 6.1775 5.1775 0.0000Staphylococcus 30 seconds 6.2405 0.9309 0.0000 aureus  1 minute  6.24051.6173 0.0000 (ATCC #29213)  3 minutes 6.2405 3.8091 0.0460  5 minutes6.2405 5.2405 0.0139  7 minutes 6.2405 5.2405 0.0000  9 minutes 6.24055.2405 0.0113 11 minutes 6.2405 5.2405 0.0283 13 minutes 6.2405 5.24050.0000 15 minutes 6.2405 5.2405 0.0000 20 minutes 6.2405 5.2405 0.0615Staphylococcus 30 seconds 5.6385 5.0233 0.0456 epidermidis  1 minute 5.6385 5.0233 0.0410 (ATCC #12228)  3 minutes 5.6385 5.0233 0.0715  5minutes 5.6385 5.0233 0.0888  7 minutes 5.6385 5.0233 0.0063  9 minutes5.6385 5.0233 0.0643 11 minutes 5.6385 5.0233 0.0211 13 minutes 5.63855.0233 0.1121 15 minutes 5.6385 5.0233 0.0321 20 minutes 5.6385 5.02330.1042

The results of this comparative In-Vitro Time-Kill evaluationdemonstrate that Microcyn oxidative reductive potential water not onlyexhibits comparable antimicrobial activity to HIBICLENS® againstEscherichia coli (ATCC #11229 and ATCC #25922), Pseudomonas aeruginosa(ATCC #15442 and ATCC #27853), Serratia marcescens (ATCC #14756), andMicrococcus luteus (ATCC #7468), but provides more effective treatmentagainst Enterococcus faecalis (ATCC #29212), Staphylococcus aureus (ATCC#6538 and ATCC #29213), and Staphylococcus epidermidis (ATCC #12228). Asshown in Table 9, Microcyn exemplifies a more rapid antimicrobialresponse (i.e., less than 30 seconds) in some species. Moreover,exposure to Microcyn results in a greater overall microbial reduction inall species listed in Table 9.

EXAMPLE 31

This example demonstrates the effectiveness of an ORP water solutionagainst Penicillin Resistant Streptococcus pneumoniae (ATCC 51915).

A culture of Streptococcus pneumoniae was prepared by using a frozenculture to inoculate multiple BAP plates and incubating for 2-3 days at35-37° C. with CO2. Following incubation 3-7 mL of sterilediluent/medium was transferred to each agar plate and swabbed to suspendthe organism. The suspensions from all plates were collected andtransferred to a sterile tube and compared to a 4.0 McFarland Standard.The suspension was filtered through sterile gauze and vortex mixed priorto use in the testing procedure.

An inoculum of 0.1 ml of the organism suspension was added to 49.9 ml ofthe Microcyn or control substance. At each exposure period, the testmixture was mixed by swirling. The test mixture was exposed for 15seconds, 30 seconds, 60 seconds, 120 seconds, 5 minutes, and 15 minutesat 25.0° C.

A 1.0 ml sample was removed from the test mixture and added to 9.0 ml ofneutralizer representing a 100 dilution of the neutralized inoculatedtest mixture. A 5 ml aliquot of the 100 neutralized inoculated testmixture was transferred to a 0.45 microliter filter apparatus pre-wettedwith 10 ml of Butterfield's Buffer. The filter was rinsed withapproximately 50 mL of Butterfield's Buffer, asepticaliy removed fromthe apparatus, and transferred to a BAP plate. Additional 1:10 serialdilutions were prepared and one (1.0) ml aliquots of the 10-3-10-4dilutions of neutralized inoculated test mixture were plated induplicate on BAP.

The bacterial subculture plates were incubated for 48±4 hours at 35-37°C. in C02. Subculture plates were refrigerated for two days at 2-8° C.prior to examination. Following incubation and storage, the agar plateswere observed visually for the presence of growth. The colony formingunits were enumerated and the number of survivors at each exposure timewas determined. Representative subcultures demonstrating growth wereappropriately examined for confirmation of the test organisms.

The exemplary ORP water solution, Microcyn, demonstrated a >99.93197279%reduction of Penicillin Resistant Streptococcus pneumoniae (ATCC 51915)after 15 second, 30 second, 60 second, 120 second, 5 minute, and 15minute contact times at 25.0° C.

EXAMPLE 32

The objective of this Example is to determine the microbial activity ofan exemplary ORP water solution (Dermacyn) versus Bacitracin using abacterial suspension assay.

Dermacyn is a ready to use product, therefore performing dilutionsduring testing was not required. Bacitracin is a concentratedre-hydrated solution requiring a dilution to 33 Units/ml.

A purchased spore suspension of B. atropheus at 2.5×107/ml was used fortesting. In addition fresh suspensions of Pseudomonas aeruginosa, andStaphylococcus aureus were prepared and measured using aspectrophotometer to ensure the titer was acceptable

Nine microliters of test substance was added to 100 ul of microbesuspension. The test mixture was held at 20° C. for the contact times of20 seconds, 5 minutes, and 20 minutes. 1.0 ml of the test mixture(entire mixture) was added to 9.0 ml of neutralizer for 20 minutes (thisis the original neutralization tube or ONT) 1.0 ml of the neutralizedtest mixture was plated on Tryptic Soy Agar in duplicate for the 5minute and 20 minute contact times. Additional dilutions and spreadplates were used for the 20 second time point, to achieve countableplates.

All plates were incubated at 30° C.-35° C. for a total of 3 days andwere evaluated after each day of incubation. To determine the number ofmicrobes exposed to Dermacyn and Bacitracin during testing thesuspensions Four 10-fold dilutions were performed and 1.0 ml of thefinal 2 dilutions was plated in duplicate, where applicable.

Dermacyn when challenged with the test organisms showed totaleradication (>4 log reduction) of the vegetative bacteria at all timepoints and for spores at the 5, and 20 minute time points. Bacitracinonly produced approximately 1 log reduction. Microcyn at the 20 secondtime point showed some reduction in spores. Bacitracin showed noevidence of lowering the bacterial or spore populations over the timeperiods tested.

EXAMPLE 33

This example demonstrates the effectiveness of two exemplary ORP watersolutions (M1 and M2) against bacteria in biofilms.

The parental strain for all studies is P. aeruginosa PAO1. Allplanktonic strains were grown aerobically in minimal medium (2.56 gNa₂HPO₄, 2.08 g KH₂PO₄, 1.0 g NH₄Cl, 0.04 g CaCl₂.2H₂O, 0.5 gMgSO₄.7H₂O, 0.1 mg CuSO₄.5H₂O, 0.1 mg ZnSO₄.H₂O, 0.1 mg FeSO₄.7H₂O, and0.004 mg MnCl₂.4H₂O per liter, pH 7.2) at 22° C. in shake flasks at 220rpm. Biofilms were grown as described below at 22° C. in minimal medium.Glutamate (130 mg/liter) was used as the sole carbon source.

Biofilms were grown as described previously (Sauer et. al., J.Bacteriol. 184:1140-1154 (2002), which is hereby incorporated byreference). Briefly, the interior surfaces of silicone tubing of aonce-through continuous flow tube reactor system were used to cultivatebiofilms at 22° C. Biofilms were harvested after 3 days (maturation-1stage), 6 days (maturation-2 stage), and 9 days (dispersion stage) ofgrowth under flowing conditions. Biofilm cells were harvested from theinterior surface by pinching the tube along its entire length, resultingin extrusion of the cell material from the lumen. The resulting cellpaste was collected on ice. Prior to sampling, the bulk liquid waspurged from the tubing to prevent interference from detached, planktoniccells.

The population size of planktonic and biofilm cells was determined bythe number of CFU by using serial dilution plate counts. To do so,biofilms were harvested from the interior surface after various periodsof time of exposure to SOSs. Images of biofilms grown in once-throughflow cells were viewed by transmitted light with an Olympus BX60microscope (Olympus, Melville, N.Y.) and a_100 magnification A100PLobjective lens. Images were captured using a Magnafire cooled three-chipcharge-coupled device camera (Optronics Inc., Galena, Calif.) and a30-ms exposure. In addition, confocal scanning laser microscopy wasperformed with an LSM 510 Meta inverted microscope (Zeiss, Heidelberg,Germany). Images were obtained with a LD-Apochrome 40_/0.6 lens and withthe LSM 510 Meta software (Zeiss).

A 2-log reduction was observed for M1-treated biofilms within 60 min oftreatment. The finding indicates that every 10.8 min (+/−2.8 min),treatment with M1 results in a 50% reduction in biofilm viability.

TABLE 10 M1 Killing. Time (min) Viability (%) 0 100 10 50 20 25 34 12.547 6.25 54 3.125

However, overall M2 was somewhat more effective in killing biofilms thanM1 because the results indicated that every 4.0 min (+/−1.2 min),treatment with M2 results in a 50% reduction in biofilm viability.

TABLE 11 M2 Killing. Time (min) Viability (%) 0 100 2.5 50 7 25 12 12.515 6.25 20 3.125

Thus, ORP water is effective against bacteria in bioflims.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A method of preventing or treating atopic dermatitis in a patient,the method consisting of administering to the patient a therapeuticallyeffective amount of an oxidative reductive potential water solution,wherein the solution has a pH of from about 6.4 to about 7.8, whereinthe oxidative reductive potential water solution comprises free chlorinespecies at a level of about 10 ppm to about 400 ppm, wherein the freechlorine species comprises hypochlorous acid, hypochlorite ions, sodiumhypochlorite and mixtures thereof, wherein the oxidative reductivepotential water solution inhibits mast cell degranulation, therebytreating the atopic dermatitis, wherein the oxidative reductivepotential water solution does not contain an antibiotic.
 2. The methodof claim 1, wherein the oxidative reductive potential water solution isadministered topically to cutaneous tissue or subcutaneous tissue. 3.The method of claim 1, wherein the oxidative reductive potential watersolution is administered as a liquid, steam, aerosol, mist or spray. 4.The method of claim 1, wherein the oxidative reductive potential watersolution is administered by aerosolization, nebulization or atomization.5. The method of claim 1, wherein the atopic dermatitis is caused by anautoimmune reaction.
 6. The method of claim 1, wherein the atopicdermatitis is caused by an infection.
 7. The method of claim 6, whereinthe infection is by one or more microorganisms selected from the groupconsisting of viruses, bacteria, and fungi.
 8. The method of claim 1,wherein the oxidative reductive potential water solution is stable forat least six months.
 9. The method of claim 1, wherein the oxidativereductive potential water solution is stable for at least one year. 10.The method of claim 1, wherein the pH of the oxidative reductivepotential water solution is from about 7.4 to about 7.6.
 11. The methodof claim 1, wherein the oxidative reductive potential water solutioncomprises from about 10% by volume to about 50% by volume of cathodewater and from about 50% by volume to about 90% by volume of anodewater.
 12. The method of claim 1, wherein the oxidative reductivepotential water solution comprises from about 15 ppm to about 35 ppmhypochlorous acid, from about 25 ppm to about 50 ppm sodiumhypochlorite, a pH of from about 6.2 to about 7.8, and the solution isstable for at least two months.
 13. The method of claim 1, wherein theoxidative reductive potential water solution further comprises chlorideion.
 14. The method of claim 1, wherein the oxidative reductivepotential water solution has a potential between about −400 mV and about+1300 mV.